Role of EMMPRIN and MMPs in tooth development, dental ...

UNIVERSITE PARIS DESCARTES

Ecole doctorale Génétique Cellule Immunologie Infectiologie

Développement

Laboratoire EA2496

Pathologies, Imagerie, et Biotherapies Orofaciales

Role of EMMPRIN and MMPs in tooth

development, dental caries and pulp-dentin

regeneration

Par Mayssam KHADDAM

THESE

Pour obtenir le grade de

DOCTEUR

Spécialité : Sciences de la Vie et de la Santé

Dirigée par Professeur Catherine CHAUSSAIN

Présentée et soutenue publiquement le 24 novembre 2014

Devant un jury composé de :

Pr. BERDAL Ariane, Université Paris Diderot Président

Pr. CHAUSSAIN Catherine, Université Paris Descartes Directeur

Pr. MOURAH Samia, Université Paris Diderot Rapporteur

Pr. MANZANARES Maria Cristina, Université de Barcelone Rapporteur

Dr. MENASHI Suzanne, Université Paris est Créteil Examinateur

Dr. ROCHEFORT Gael, Université Paris Descartes Examinateur

Dr. HUET Eric, Université Paris est Créteil invité

UNIVERSITE PARIS DESCARTES

Ecole doctorale Génétique Cellule Immunologie Infectiologie

Développement

Laboratoire EA2496

Pathologies, Imagerie, et Biotherapies Orofaciales

Role of EMMPRIN and MMPs in tooth

development, dental caries and pulp-dentin

regeneration

Par Mayssam KHADDAM

THESE

Pour obtenir le grade de

DOCTEUR

Spécialité : Sciences de la Vie et de la Santé

Dirigée par Professeur Catherine CHAUSSAIN

Présentée et soutenue publiquement le 24 novembre 2014

Devant un jury composé de :

Pr. BERDAL Ariane, Université Paris Diderot Président

Pr. CHAUSSAIN Catherine, Université Paris Descartes Directeur

Pr. MOURAH Samia, Université Paris Diderot Rapporteur

Pr. MANZANARES Maria Cristina, Université de Barcelone Rapporteur

Dr. MENASHI Suzanne, Université Paris est Créteil Examinateur

Dr. ROCHEFORT Gael, Université Paris Descartes Examinateur

Dr. HUET Eric, Université Paris est Créteil invité

Acknowledgment:

At the end of my thesis I would like to thank all those people who made this thesis possible

and an unforgettable experience for me.

First of all, I would like to express my deepest sense of gratitude to my supervisor Prof.

Catherine Chaussain who offered her continuous advices and encouragement throughout

the course of this thesis. I thank her for the systematic guidance and great effort she put into

training me in the scientific field.

I am very grateful to Prof. Samia Mourah, from Paris Diderot University and Prof. Maria

Cristina Manzanares, from Barcelona University for being part of this PhD thesis

committee and for accepting being the reviewers of this PhD thesis.

I would like to thank Prof. Ariane Berdal, from Paris Diderot University, Dr. Suzanne

Menashi from Paris Est Créteil University, Dr. Gael Rochefort from Paris Descartes

University, and Dr. Eric Huet from Paris Est Créteil University for accepting to be members

of this PhD thesis committee. I am grateful and honored by their participation.

I would like to thank Dominique Le-Denmat, Dominique Septier, and Brigitte Baroukh; I

learned a lot of things from your scientific and general experiences.

Thanks to Tchilalo Boukpessi; TP at 8:00 Am was a good chance for me to catch some of

your experience in teaching, and i am happy to work with you in GSE article.

Thanks to Elvire Le Norcy; 6 months in the orthodontics department at Charles Foix (Ivry-

sur-Seine) hospital was very useful for me especially with your experience.

I would like to thank Anne Polliard, Claire Bardet, Sandy Ribes, Julie Lesieur, Benjamin

Salmon, Jeremy Sadoine, Benoît Vallée, Sibylle Vital, Céline Gaucher, Marjolaine Gosset,

Bernard Pellat, Jean-Louis Saffar, Marie-Laure Colombier, and Philippe Bouchard.

Cedric Mauprivez and Caroline Gorin, we are at the end of our thesis so I hope that

everything will be OK.

Thanks to Elisabeth Jimenez for her help in all my administrative problems.

I am thankful to all in EA 2496: Anne-Margaux Collignon, Benjamin Coyac, Maxime

Ghighi, Frederic Chamieh, Laurent Detzen, Jean-Baptiste Souron, Annie Llorens, Aurélien

Vernon, Cyril Willig, Maheva Garcia, Anita Novais, Sonia Pezet, Tania Selbonne, and

Alexandra Benoist.

Special thanks to Jiyar Naji, Xuan Vinh Tran, Soledad Acuña Mendosa, Francesca Mangione,

Yong Wu, and Bassam Hassan; you are completely different and I am so happy to work with

you.

Thanks to Jotyar Khlaf Arif for his help.

I am thankful to Syrian government who gives me the grant to continue my study in France.

These acknowledgments would not be complete without thanking my family for their

constant support and care. Very special thanks to my parents, despite they can’t read these

words but they know how i appreciate their efforts and their times spent for me.

Finally, I would like to mention two other people who are very important in my life: My

wife, Khawla and my little daughter, Nada. I thank Khawla for her encouragement and

support, especially in the hardest time. I thank my little daughter for making me so happy

with her cute smile which gives me the hope at the most difficult moments.

1

Table of Contents Table of figures ....................................................................................................................................... 2

List of abbreviations ................................................................................................................................ 6

General introduction and specific objectives ......................................................................................... 8

1 Introduction .................................................................................................................................. 11

1.1 Tooth description .................................................................................................................. 11

1.2 Tooth development .............................................................................................................. 12

1.2.1 Stages of tooth development ....................................................................................... 13

1.2.2 Basement membrane .................................................................................................... 17

1.2.3 Dentin ............................................................................................................................ 19

1.2.4 Enamel .......................................................................................................................... 23

1.3 Matrix MetalloProteinases MMPs ........................................................................................ 30

1.3.1 MMPs and teeth ........................................................................................................... 31

1.4 EMMPRIN (Basigin,CD147).................................................................................................... 32

1.4.1 Historic .......................................................................................................................... 32

1.4.2 Structure ....................................................................................................................... 32

1.4.3 Phenotypes of EMMPRIN knock out (KO) mice ............................................................ 35

1.4.4 EMMPRIN interactions .................................................................................................. 35

5.4.1 EMMPRIN functions ...................................................................................................... 37

6.5.4 EMMPRIN and tooth ..................................................................................................... 43

2 MMPs and dentin matrix degradation .......................................................................................... 48

3 Results ........................................................................................................................................... 57

3.1 Role of EMMPRIN in tooth formation ................................................................................... 57

3.1.1 Supplementary data ...................................................................................................... 69

3.1.2 Supplementary results .................................................................................................. 72

3.2 Role of EMMPRIN in pulp-dentin regeneration .................................................................... 79

3.2.1 Background and project aim ......................................................................................... 79

3.2.2 Materials and methods ................................................................................................. 79

3.2.3 Results ........................................................................................................................... 80

3.3 Inhibition of MMP-3 and dentin matrix degradation ........................................................... 82

4 Discussion ...................................................................................................................................... 91

5 References .................................................................................................................................... 95

6 Annexe 1 ..................................................................................................................................... 123

2

Table of figures

Figure 1 : The tooth and its supporting structure. Adapted from (Antonio Nanci and Cate

2013) ........................................................................................................................................ 11

Figure 2 : Adult mouse mandible (own data). ........................................................................ 12

Figure 3 : Dental lamina of tooth development. Adapted from (Antonio Nanci and Cate

2013) ........................................................................................................................................ 13

Figure 4 : Bud stage of tooth development. Adapted from (Antonio Nanci and Cate 2013) . 14

Figure 5 : Cap stage tooth germ showing the position of the enamel knot. Adapted from

(Antonio Nanci and Cate 2013) ............................................................................................... 15

Figure 6 : Cap stage, beginning of cellular differentiation within the enamel organ. Central

cells form the stellate reticulum. Adapted from (Antonio Nanci and Cate 2013) ................... 16

Figure 7 : Early bell stage of tooth development (own data) .................................................. 17

Figure 8 : Characteristics of dentin formation. Odontoblasts secrete an ECM composed of

type I collagen and NCPs. Within the predentin type I collagen molecules are assembled as

fibrils. Mineralization occurs at the mineralization front by growth and fusion of

calcospherites formed by hydroxyapatite (HAP) crystals. This mineralization process is

controlled by NCPs and by mineral ion availability. Cell processes remain entrapped within

dentin whereas cell bodies remain at the periphery of the pulp. Adapted from (Vital et al.

2012) ........................................................................................................................................ 20

Figure 9 : Human dentin by scanning electronic microscopy (SEM). A. cutting line is

parallel to dentin tubules, B. cutting line is perpendicular to dentin tubules. PtD: peritubular

dentin, ItD: intertubular dentin. (Own data) ............................................................................ 21

Figure 10 : Scanning electron microscope views of (A) the enamel layer covering coronal

dentin, (B) the complex distribution of enamel rods across the layer, (C and D) and

perspectives of the rod-interrod relationship when rods are exposed longitudinally (C) or in

cross section (D). Interrod enamel surrounds each rod. DEJ: Dentinoenamel junction; IR:

interrod; R,rod. Adapted from (Antonio Nanci and Cate 2013) .............................................. 24

Figure 11 : Semi-thin (0.5 µm) sections from glutaraldehyde-fixed, decalcified, and plastic

embedded mandibular incisors of wild-type mice stained with toluidine blue to illustrate the

appearance of enamel and enamel organ cells at mid-secretory stage (A) and near-mid-

maturation stage (B) of enamel development. Abbreviations: E, enamel; Am, ameloblast; Si,

stratum intermedium; pd, predentin; D, dentin; ae, apical end; be, basal end; bv, blood vessel;

as, artifact space; b, bone; c, cementum. Adapted from (J. D. Bartlett and Smith 2013) ........ 29

Figure 12 : Schema present incisor enamel and denin formation. p-Am: pre-ameloblast; pOd:

pre-odontoblast; s-Am: secreting Ameloblasts; od: odontoblasts; pos-Am: post-secretory

ameloblasts; pD: predentin; D: dentin; pm-E: premature enamel; E: enamel. Adapted from

(Khaddam et al. 2014).............................................................................................................. 30

Figure 13 : Schematic representation of MMP activity during the dentin carious process.

Cariogenic bacteria present in the caries cavity release acids such as lactic acid that reduce

the local pH. The resulting acidic environment demineralizes the dentin matrix and induces

the activation of host MMPs derived from dentin or saliva (which bathes the caries cavity).

Once the local pH is neutralized by salivary buffer systems, activated MMPs degrade the

demineralized dentin matrix. Adapted from (Chaussain et al. 2013) ...................................... 32

3

Figure 14: Basigin isoforms. Characteristic features of isoforms are mentioned within

blanket. Carbohydrates are shown by light grey color. Adapted from (Takashi Muramatsu

2012) ........................................................................................................................................ 33

Figure 15: Scheme of EMMPRIN structure. EMMPRIN contains an extracellular domain

composed of two Ig loops with three Asn-linked oligosaccharides and short single

transmembrane domain (TM) and a cytoplasmic domain (Cyt). The first Ig domain is required

for counter-receptor activity, involved in MMP induction. Adapted from (Gabison et al.

2009). ....................................................................................................................................... 34

Figure 16: Possible EMMPRIN-mediated interactions stimulating MMP production. (A)

Homophilic cis interaction between EMMPRIN molecules within the plasma membrane of a

tumor cell. (B) Homophilic trans interaction between EMMPRIN molecules on apposing

tumor cells. (C) Heterophilic interactions between EMMPRIN on a tumor cell and a putative

EMMPRIN receptor on a fibroblast. Adapted from (Toole 2003)........................................... 35

Figure 17 : Tumor-cell induced activation of adjacent fibroblasts by homophilic EMMPRIN

signaling. Adapted from (Joghetaei et al. 2013) ...................................................................... 40

Figure 18 : Immunoreactivity (IR) for EMMPRIN. a Cells of the inner enamel epithelium

(cap stage of the enamel organ) show intense EMMPRIN IR (Alexa-coupled). b Ameloblasts

as well as odontoblasts (bell stage of the enamel organ) exhibit strong EMMPRIN IR. Note

the IR in the borderline between ameloblasts and the stratum intermedium. Mesenchymal

cells of the dental papilla are only weakly immunoreactive. Abbreviations: A ameloblast; DL

dental lamina; EEE external enamel epithelium; IEE internal enamel epithelium; EO enamel

organ; Od odontoblast; SI stratum intermedium; SR stellate reticulum. Adapted from

(Schwab et al. 2007) ................................................................................................................ 43

Figure 19 : Transcription level of EMMPRIN in different stages of tooth development. a

EMMPRIN mRNA was higher in E13.0 mandible than that in E11.0. b The expression of

EMMPRIN mRNA was higher in P1 tooth germ than that in E14.0. Adapted from (Xie et al.

2010) ........................................................................................................................................ 44

Figure 20 : Examination the role of EMMRIN in early tooth germ development using

EMMPRIN siRNA in the cultured mandible at E11.0. a After being cultured for 6 days, the

tooth germ was found to have developed into the cap stage in mandibles cultured with

scramble siRNA. b Dental epithelial bud was observed in the mandible treated with

EMMPRIN siRNA after 6 days of culture. c A cap-like mature enamel organ was observed in

the mandibles with scrambled siRNA supplement at 8-day culture. d EMMPRIN

siRNAtreated mandible explants also showed a bud-like tooth germ at 8-day culture.

EMMPRIN siRNA had a specific effect on the morphogenesis of tooth germ. DE dental

epithelium, DM dental mesenchyme, DP dental papilla, EO enamel organ, OE oral

epithelium, PEK primary enamel knot. Adapted from (Xie et al. 2010) ................................. 45

Figure 21 : Temporal expression and localization of EMMPRIN in the gingival epithelium

during ligature-induced periodontitis in the first mandibular molar of rats. (A) On day 0

(health), the immunoreactivity was strong in the basal cells, with a decrease toward the upper

layers in the attached gingival epithelium (star in a1). (B) On day 7, immunoreactivity was

greatly enhanced in the attached gingiva (star in b1). (C) On day 15, immunoreactivity was

4

similar to that seen in the healthy state in the attached gingival epithelium (star in c1).

Adapted from (L. Liu et al. 2010) ............................................................................................ 46

Figure 22 : EMMPRIN expression in the developing incisor of 3 month-old mice

Immunostaining with EMMPRIN antibody on sagittal section of the mandible shows that the

secretory ameloblasts, the stratum intermedium and odontoblasts are positive for EMMPRIN

(A and B). By contrast, no staining is observed in the post-secretory ameloblast (C). Am:

ameloblast; s-Am: secretory ameloblast; pos-Am: post-secretory ameloblast; Od: odontoblast;

D: dentin; pD: predentin; pm-E: premature enamel; Si: stratum intermedium; fm: forming

matrix. From (Khaddam et al. 2014) ....................................................................................... 69

Figure 23 : KLK-4 expression in tooth germs of EMMPRIN KO mice when compared with

WT. For mRNA expression, a 33 % increase is observed by qRT-PCR in KO mice. KLK-4

activity is hardly detectable by casein zymography (with 20 mM EDTA in the incubation

buffer to inhibit MMP activity). No activity is seen for recombinant MMP-20. From

(Khaddam et al. 2014).............................................................................................................. 70

Figure 24 : SEM observation of 3 month-old mouse mandible sections. At M1 level, no

difference in the morphology of either the bone or the teeth is detected between WT and KO

mice (A, B). Both dentin (E, F) and enamel appear normal and the enamel prisms are

normally constituted (C, D). From (Khaddam et al. 2014). .................................................... 71

Figure 25: TEM analysis was performed on M1 and M2 germs of new born mice. In the KO

M2 germs, a cell polarization delay is observed in both pre-ameloblasts and pre-odontoblasts

localized at the tip of the cusps (b). In the WT, well-organized ameloblast and odontoblast

palisades are seen, with a basal localization of the nuclei and long cell processes (arrow-

heads) (a), whereas in the KO, cells are seen proliferating with centrally localized nucleus

(b). At higher magnification, the basement membrane (black arrows) is partially degraded in

WT (white arrows) (c), but appears still intact in the KO (d). In M1germ, the basement

membrane which can no longer be detected in the WT (e) is partially degraded in the KO

(arrow) (f). Dentin matrix (black arrow-heads) is secreted in both mice models (e-f-g-h) but

at a higher rate in the WT (e) where a greater amount of fibrillated collagen is seen associated

with hydroxyapatite crystals (white arrow heads). In addition, mineralizing enamel matrix

can already be observed at the secreting pole of WT ameloblasts localized at the tip of the

cusp (g) but is not detectable in the KO (h). pam: pre-ameloblast; pod: pre-odontoblast; am:

ameloblast; od: odontoblast; fde: forming dentin; fen: forming enamel. (own data). ............. 73

Figure 26: EMMPRIN expression in the first molar of mouse embryo. immunoreactivity (IR)

for EMMPRIN in paraffin sections of mouse embryo tooth germ tissue at 16 day and 17 day

(cap stage). Inner enamel epithelium cells show EMMPRIN IR, this IR in the buccal side is

stronger than in the lingual side of the molar germ. Iee: innerenamel epithelium; dp: dental

pulp; bs: buccal side; ls: lingual side. (own data) .................................................................... 74

Figure 27: Alveolar bone density. ........................................................................................... 76

Figure 28: Percent of bone volume in VOI............................................................................. 76

Figure 29: Trabecular bone thickness in VOI. ........................................................................ 77

Figure 30: Trabecular number in VOI. ................................................................................... 77

Figure 31: Trabecular separation in VOI. ............................................................................... 78

5

Figure 32: Mouse first upper molar after 7 and 28 days of capping with Biodentine. 7 days

post operatively, dentin formation was detected in +/+ and -/- EMMPRIN mice (A,C), but it

was more in -/- (brown arrow C) than in +/+ (brown arrow A). 28 days post operatively,

dentine bridge was visible, but it was more continuous in -/- (arrow in D) than in +/+ (arrow

in B) where it was not continued. e: enamel; d: dentin; GIC: glass ionomer cement; red *:

Biodentine. ............................................................................................................................... 80

Figure 33: Percent of dentin volume in volume of interest VOI. Significant increase in dentin

density was detected in -/- EMMPRIN mice when compared with +/+ mice at 7 days post

operatively................................................................................................................................ 81

Figure 34: Recapitulative schema proposing the role of EMMPRIN in tooth formation. At

early bell stage, EMMPRIN is expressed by pre-ameloblast (p-Am) and may orchestrate

basement membrane degradation (black line) to allow direct contact with pre-odontoblast (p-

Od), which is mandatory for the final cell differentiation. At secretory stage, both secreting

ameloblasts (s-Am) and odontoblasts (Od) highly express EMMPRIN. This expression may

enhance MMP-20 synthesis by ameloblasts allowing for early enamel maturation. At the

enamel maturation stage, post-secretory ameloblasts (pos-Am) lose their EMMPRIN

expression. The arrows indicate EMMPRIN expression by cells. The red line schematizes the

time window where a direct effect of EMMPRIN is allowed by a direct cell contact. D:

dentin; pD: predentin; pm-E: premature enamel; E: enamel. .................................................. 92

6

List of abbreviations

AI: Amelogenesis imperfecta

Ambn : Ameloblastin

AMTN : Amelotin

Asn : Asparagine

ATK: serine/threonine kinase and is known as protein kinase B (PKB) or RAC-PK (‘related

to A and C protein kinase’)

ASARM: acidic serine and aspartate-rich motif

BM: Basement membrane

BSP : Bone sialoprotein

BV : Bone volume

CyPA : Cyclophilin A

DI : Dentinogenesis imperfecta

DMP-1 : Dentin matrix protein 1

DSPP : Dentin sialophosphoprotein

DEJ: Dental enamel junction

ECM : Extra cellular matrix

Enam : Enamelin

EMMPRIN : Extra cellular matrix metalloproteinase inducer

GAG: Glycoaminoglycans

GIC: Glass ionomer cement

HAP : Hydroxyapatite crystals

HIF-1α : hypoxia-inducible factor 1α

KLK-4 : Kallikrine-4

KO: Knock out

MAPK: Mitogen-activated protein

MCT : Monocarboxylate transporter

MEPE: Matrix extracellular phosphoglycoprotein

MMP : Matrix metalloproteinase

MMP-1 : Collagenase 1

MMP-2 : Gelatinase A

MMP-3 : Stromelysin 1

MMP-9 : Gelatinase B

MMP-20 : Enamelysin

7

MVs : Membrane vesicules

NCPs: Non-collagenous proteins

OPN : Osteopontin

PDL : Periodontal ligament

PFA: Para-formaldehyde

PG: Proteoglycans

PI3K: Phosphoinositide 3-kinase

RGD: Arginine–glycine–aspartate cell adhesion domain

RA: Rheumatoid arthritis

SEM: Scanning electron microscopy

SIBLINGs : Small integrin-binding ligand N-linked glycoprotein

SLRPs: Small leucine-rich proteoglycans

Tb: Trabecular bone

TEM: Transmission electron microscopy

TIMP: Tissue inhibitor of MMPs

TV: Tissue volume

VEGF: Vascular endothelial growth factor

VOI : Volume of interest

8

General introduction and specific objectives

Tooth development results from reciprocal inductive interactions between the

ectomesenchyme and oral epithelium and proceeds through a series of well-defined stages

including the initiation, bud, cap and bell stages (Ruch, Karcher-Djuricic, and Gerber 1973;

Slavkin 1974; Catón and Tucker 2009; Miletich and Sharpe 2003; I Thesleff and Hurmerinta

1981; Mitsiadis and Luder 2011). At the bell stage which is the last step of tooth crown

formation, signals from the dental epithelium (i.e., inner enamel epithelium) instruct dental

mesenchymal cells to differentiate into odontoblasts. Differentiated odontoblasts signal back

to inner enamel epithelial cells and induce their differentiation into ameloblasts, which are

responsible for enamel matrix synthesis. Ameloblast terminal differentiation necessitates the

presence of an extracellular matrix that is secreted by odontoblasts and forms the predentin

(Zeichner-David et al. 1995). The degradation of the basement membrane (BM) separating

the dental epithelium from the mesenchyme is a key step in this process that allows direct

contact of ameloblasts with both odontoblasts and the unmineralized dentin matrix (Catón

and Tucker 2009; Olive and Ruch 1982). Matrix metalloproteinases (MMPs) are involved in

all stages of tooth formation (Bourd-Boittin et al. 2005; Chaussain-Miller et al. 2006). At the

bell stage, MMPs have a major role in BM degradation (Heikinheimo and Salo 1995;

Sahlberg et al. 1992a), thus allowing direct cross-talk between odontoblasts and ameloblasts

(Heikinheimo and Salo 1995; Sahlberg et al. 1999). It has been shown that at more advanced

stages MMPs also regulate the processing of dental extracellular matrix (ECM) proteins prior

to mineralization. Indeed, it has been demonstrated that MMPs regulate amelogenin (AMEL)

cleavage by enamelysin (MMP-20) during early enamel maturation (Bourd-Boittin et al.

2005; Bourd-Boittin et al. 2004; Lu et al. 2008; Nagano et al. 2009; Turk et al. 2006; Simmer

and Hu 2002; J. D. Bartlett and Simmer 1999). The notion of direct epithelial-mesenchymal

(or epithelio-stromal) interactions was first introduced in the cancer field when EMMPRIN, a

membrane glycoprotein also known as CD147, was identified as a MMP inducer present at

the cell surface of tumor cells which can activate stromal cells through direct contact and

signal them to increase MMP production (Toole 2003). Recently accumulating data also

advocate a role for EMMPRIN in modulating MMP expression during non-tumorigenic

pathological conditions as well as in physiological situations such as tissue remodeling and

cytodifferentiation events (Gabison, Hoang-Xuan, et al. 2005; Huet, Gabison, et al. 2008;

Mohamed et al. 2011; Kato et al. 2011; Nabeshima et al. 2006; L. Liu et al. 2010; Gabison et

9

al. 2009; Zhu et al. 2014) . The expression of EMMPRIN in the developing tooth germs was

previously described (Schwab et al. 2007; Xie et al. 2010). EMMPRIN expression was shown

to increase gradually in the forming molar germ in mice from E14 to P1(Xie et al. 2010).

However, the in vivo role of EMMPRIN in tooth development and homeostasis is still

unknown. In this PhD, our first specific objective was to investigate EMMPRIN

functions in tooth formation using EMMPRIN KO mice by exploring the modifications

occurring in their dental phenotype and the consequences on EMMPRIN’s molecular targets,

in particular on MMPs.

In parallel, EMMPRIN has been shown to be involved in the repair process of

different injured tissues. Indeed, the role of EMMPRIN in wound healing through MMP

induction and increase in myofibroblast contractile activity has been established (Gabison,

Mourah, et al. 2005; Huet, Vallée, et al. 2008). As our team has developed several pulp injury

models to follow-up the repair process, and as we had access to EMMPRIN KO mice it was

tempting to study the repair process in this model. Therefore, our second specific objective

was to investigate for a potential role of EMMPRIN in the pulp dentin repair process by

comparing the healing of injured pulps of EMMPRIN KO and WT mice.

MMPs were shown to be expressed during tooth development and to be necessary for

normal dentin formation (Bourd-Boittin et al. 2005). After dentin mineralization, they remain

trapped in the calcified matrix either under active or proenzyme forms (Palosaari et al. 2003),

which may explain their persistent presence within the dentin of adult teeth (A Mazzoni

2007; Tjäderhane et al. 1998). The role of these trapped MMPs in the progression of the

carious process within dentin matrix has been proposed by several studies (Tjäderhane et al.

1998; Sulkala et al. 2001). Indeed, MMPs have been proposed to have an important role in

the dentin organic matrix degradation following demineralization by bacterial acids

(Tjäderhane et al. 1998; Chaussain-Miller et al. 2006). Cariogenic bacteria are essential to

initiate the carious process but they cannot degrade the dentin organic matrix. After

dissolution of the mineral part, the organic part of dentin becomes exposed to degradation by

host-derived enzymes, including salivary and dentinal MMPs, and cysteine cathepsins

(Nascimento et al. 2011; van Strijp et al. 2003). Because MMPs have been suggested to

contribute to dentin caries progression, the hypothesis that MMP inhibition would affect

dentin caries progression is appealing. This hypothesis was supported by in vivo studies in rat

caries models where dentin caries progression was delayed by intra-oral administration of

chemical MMP inhibitors, modified tetracylines and zoledronate (Sulkala et al. 2001;

10

Tjäderhane et al. 1999). Several natural molecules have been previously reported to have

MMP inhibitory properties. Grape-seed extracts (GSE) have been shown to suppress

lipopolysaccharide-induced MMP secretion by macrophages and to inhibit MMP-1 and

MMP-9 activities in periodontitis (La et al. 2009). The MMP-inhibitory effects of these

natural substances suggest, therefore, that they could be effective in inhibiting dentin caries

progression. Recently, a new daily mouthrinse composed of grape-seed extracts and amine

fluoride has been developed. As grape-seed extracts are known to be natural inhibitors of

MMPs, our last specific objective was to evaluate the capacity of these natural agents to

prevent the degradation of demineralized dentin matrix by MMP-3.

11

1 Introduction

1.1 Tooth description

Tooth is the hardest organ of the mammalian body and it provides several functions such as

mastication, and phonation.

Anatomically, tooth structure can be distinguished in a visible part (crown) and a hidden part

embedded in the alveolar bone of the jaw (root) (Figure 1). Instead of a considerably

different shape and size (e.g., an incisor compared with a molar), teeth are histologically

similar.

Figure 1 : The tooth and its supporting structure. Adapted from (Antonio Nanci and Cate

2013)

Tooth consists of several layers: enamel, dentine, cement, and dental pulp. The enamel is a

hard, and acellular structure formed by epithelial cells and supported by dentin. This less

mineralized, more resilient, and vital hard connective tissue, is formed and supported by the

dental pulp, a soft connective tissue (Figure 1).

12

In mammals, teeth are attached to the bones of the jaw by the periodontium, consisting of the

cementum, periodontal ligament (PDL) and alveolar bone, which provide an attachment with

enough flexibility to withstand the forces of mastication.

Human and most of the mammals have two generations of teeth, primary and permanent;

since the size of teeth cannot increase after formation, the primary dentition becomes

inadequate and must be replaced by more and larger teeth (permanent dentition).

Otherwise, mice have only one generation highly reduced dentition having one incisor,

separated from three molars by an edentulous region in each semi-maxilla (Figure 2.A).

Incisor growth is continuous throughout the animal’s life (Figure 2.B).

Figure 2 : Adult mouse mandible (own data).

1.2 Tooth development

Since toothed vertebrate have conserved tooth development process stages, data obtained

from rodents studies may provide a lot of information about dental development in diverse

species, including humans (Streelman et al. 2003).

Organogenesis results from three fundamental processes: I) initiation, within positional

information are provided and interpreted to initiate organ formation at the right place; II)

13

morphogenesis, in which cells build up a rudimental organ; finally, III) differentiation where

cells form organ-specific structures.

As also showed in mouse tooth development model (Irma Thesleff and Nieminen 1996), teeth

are vertebrate-specific structures which, like many other organs, develop through a series of

reciprocal interactions between two adjacent tissues, an epithelium and a mesenchyme (I

Thesleff, Vaahtokari, and Partanen 1995). Tissue-recombination experiments have shown

that the oral epithelium isolated from the mandibular arch of a mouse embryo, between

embryonic day 9.0 and 11.5 (E9.0–E11.5), can stimulate a non-dental neural crest-derived

mesenchyme to form a tooth. After E11.5, the odontogenic potential subsequently shifts

from the epithelium to the mesenchyme, which can induce dental formation when combined

with a non-dental epithelium, whereas the dental epithelium has lost this ability(Mina and

Kollar 1987; Lumsden 1988).

1.2.1 Stages of tooth development

Tooth development takes place through a series of well-defined stages: epithelial thickening

of the dental lamina, bud, cap and bell.

1.2.1.1 Dental lamina Stage

The thickening of the mouse oral epithelium is first visible at around E11.5 (Figure 3). The

epithelial thickening forms the dental and vestibular lamina on the lingual and buccal aspect,

respectively. The vestibular lamina forms a sulcus between the cheek and the teeth, and the

dental lamina gives rise to the teeth. During this stage, dental lamina expresses several

important signaling molecules such as (Sonic Hedgehog) Shh that increases cell proliferation

at the tooth development site (Hardcastle et al. 1998).

Figure 3 : Dental lamina of tooth development. Adapted from (Antonio Nanci and Cate

2013)

14

1.2.1.2 Bud stage

After the dental lamina stage, an epithelial structure that has a bud shape results from

proliferating and invagination of the epithelium within the underlying ectomesenchyme. The

bud is clearly formed at E13.5 and it consists in several layers: the dental follicle made by

condensed mesenchymal cells, oriented in a radial pattern and encasing the dental papilla and

the enamel organ; enamel organ, in which the internal epithelial cells meets the external

epithelial cells and form a structure called the cervical loop; finally, dental papilla, which is a

ball of densely packed ectomesenchyme (Figure 4).

Figure 4 : Bud stage of tooth development. Adapted from (Antonio Nanci and Cate 2013)

1.2.1.3 Cap stage

Around E14.5, the condensing mesenchyme signals back to the enamel organ and induces the

formation of a specific group of signaling epithelial cells known as the enamel knot which

takes control of odontogenesis processes (Irma Thesleff, Keranen, and Jernvall 2001). The

enamel knot is visible as a bulge in the center of the inner enamel epithelium at the cap stage

(Figure 5). Enamel knot expresses a host of signaling molecules, such as Shh, Fgf4, Bmp4

and Wnt10b (Vaahtokari et al. 1996; Sarkar and Sharpe 1999).

Then, in multi-cusped teeth, secondary enamel knots guides the differentiation at each cusp

tip, during the bell and crown formation stages (Irma Thesleff, Keranen, and Jernvall 2001;

Matalova et al. 2005).

15

Figure 5 : Cap stage tooth germ showing the position of the enamel knot. Adapted from

(Antonio Nanci and Cate 2013)

By E 15, the differentiation of enamel organ central cells forms the stellate reticulum cells

(Figure 6) having a star shape with large intercellular spaces potentially playing a role in

enamel-secreting ameloblasts nutrition. Another layer of cells known as “stratum

intermedium”, at E16.0 in the incisor and E17.0 in the molar, becomes recognizable from the

internal dental epithelial cells as flattened epithelial cells, between the stellate reticuIum and

the internal dental epithelium whose cells, progressively, lengthened to become

preameloblasts.

16

Figure 6 : Cap stage, beginning of cellular differentiation within the enamel organ.

Central cells form the stellate reticulum. Adapted from (Antonio Nanci and Cate 2013)

1.2.1.4 Bell stage

During this stage (EI7.0 for incisor and by E17.5-18.0 for molars), dental papilla cells

differentiate into odontoblasts, beginning in the most anterior mesenchymal cells (Figure 7).

The external dental epithelial cells thickness decreases and becomes a one or two cuboidal

cell layer.

The preameloblasts about double in height and differentiate into ameloblasts and their nuclei

peripherally placed, this differentiation firstly occurs in the most anterior regions. The lingual

side of the incisors does not become coated with enamel because that the internal dental

epithelial cells do not differentiate into ameloblasts on this side. At El7.0 these non-

differentiating internal dental epithelial cells, diminish and become cuboidal in shape in

subsequent stages of development, then merge with adjacent connective tissue cells.

By EI8 in the incisors and El9 in molars, odontoblasts begin to secrete predentin (Figure 7).

After 24 hours of development, the predentin starts mineralizing and enamel matrix will be

secreted by ameloblasts. Mineralization of the enamel matrix is postnatal and the incisors and

the first molar erupt by day 20 after birth (P20). Tooth shape will be established when

mineralization of dentin and enamel starts.

17

Figure 7 : Early bell stage of tooth development (own data)

1.2.1.5 Second and Third Molar Development

When the jaws elongate enough, the second and third molars start developing. Second molar

development starts with the dental lamina, which can be seen at E15.5 forming as an

outgrowth of the first molar tooth germ epithelium. By E18.5 the second molar is at the cap

stage and erupts approximately at P25. The lamina of the third molar appears at P4, reaches

the cap stage by P7-9 and the bell stage by P10, the third molar erupts by P35 (Rossant and

Tam 2002).

1.2.2 Basement membrane

The basement membranes (BM) are the first extracellular matrices to appear and they are

critical for organ formation and tissue repair (Martin and Timpl 1987; Kleinman et al. 1986).

They act like scaffolds for cells and play an essential role in morphogenesis that affects cell

adhesion, migration, proliferation, and differentiation.

The structure and components of BMs vary among tissues, resulting in tissue-specific

structures and functions. BMs consist of supramolecular structure which is formed by

reciprocal interaction of collagen IV, laminin, perlecan, nidogen/entactin, and other

molecules (Martin and Timpl 1987; Kleinman et al. 1986).

18

BM components play an important role in tooth development. They control proliferation,

polarity, attachment and determine tooth germ size and morphology (I. Thesleff et al. 1981;

Fukumoto and Yamada 2005; Fukumoto et al. 2006).

For example, laminin α5 (Lama5), is a component of the major laminin chain in tooth

basement membranes. Absence of Lama5 in KO mice lead to a small tooth germ with no

cusps, in which the inner dental epithelium is not polarized and enamel knot formation is

defective (Fukumoto et al. 2006).

Another laminins such as laminin α2 (Lama2) are expressed in odontoblasts during the late

stage of germ development (Yuasa et al. 2004; Salmivirta, Sorokin, and Ekblom 1997). Its

deficiency in mice manifests in thin dentin and defective dentinal tube structure (Yuasa et al.

2004). These phenotypes are similar to dentinogenesis imperfecta (DI) in humans. It was

found that laminin-2, increases dentin sialoprotein expression in odontoblasts in cell culture,

but its deficiency in mutant mice, reduces dentin sialoprotein expression in odontoblasts,

suggesting that Lama2 is required for odontoblast differentiation.

Perlecan (HSPG2) is a major heparan sulfate proteoglycan in BMs. Its expression in

developing teeth, was detected in BMs, intercellular spaces of the enamel organ, and the

dental papilla including odontoblasts (Ida-Yonemochi et al. 2005). Overexpression of

perlecan in transgenic mice results in abnormal tooth morphology and deregulation of growth

factors such as TGF-b1 and bFGF (Ida-Yonemochi et al. 2011).

19

1.2.3 Dentin

1.2.3.1 Dentin structure

Dentin has a complex structure, similar to bone for mineralization ratio of about 70%

mineral. In contrast with bone, dentin is not vascularized, and has not remodeling process.

During the secretory stage, odontoblasts polarize, elongate and start to display two distinct

parts: a cell body and a process. During the next step of evolution, the cell bodies stay outside

the mineralized dentin, along the border of the mineralization front and the processes occupy

the lumen of dentin tubules. Tubule diameter varies between 2 and 4 micrometers and its

number is about 18 000 and 21 000 tubules per mm2 (Schilke et al. 2000). They are more

numerous in the inner third layer than the outer third layer of the dentin.

1.2.3.1.1 Outer mantel dentin layer

Outer mantel dentin is a thin atubular layer with thickness of 15–30µm, at the periphery of

coronal region. It is less mineralized than the rest of dentin and consequently the resilient

mantle dentin allow dentin to dissipate pressures which otherwise would induce enamel

fissures and detachment of the fragmented enamel from the dentin-enamel junction(R. Z.

Wang and Weiner 1998).

1.2.3.1.2 Circumpulpal dentins

The circumpulpal dentin appears as a thin layer at initial stages of dentinogenesis, its

thickness continuously increases at the expense of the pulp and then it becomes the largest

part of the dentin layer. The circumpulpal dentin is formed by circles of peritubular dentin

around the lumen of the tubules separated by the intertubular dentin. The ratio between inter

and peritubular dentin is species dependent, it is about 50% in horses and about 10-20% in

humans, and in the continuously growing rodent incisors no peritubular dentin can be found.

Several differences in the structure and composition of these two types of dentin are found. In

the intertubular dentin, the major protein is type I collagen (90%), whereas in the peritubular

dentin no collagen is observed. The differences in the composition of noncollagenous

proteins (NCPs) of the two types of dentin have been reported. (M. Goldberg, Molon Noblot,

and Septier 1980; Weiner et al. 1999; Gotliv, Robach, and Veis 2006; Gotliv and Veis 2007).

Intertubular dentin (Figure 9) results from transformation of predentin into dentin (Figure 8).

It is compound of dense network of collagen fibrils, coated by NCPs, where needle like-

crystallites locate at the collagen fibrils parallel to their axes and other crystallites fill inter-

fibrillar spaces (M. Goldberg and Boskey 1996).

20

Figure 8 : Characteristics of dentin formation. Odontoblasts secrete an ECM composed

of type I collagen and NCPs. Within the predentin type I collagen molecules are

assembled as fibrils. Mineralization occurs at the mineralization front by growth and

fusion of calcospherites formed by hydroxyapatite (HAP) crystals. This mineralization

process is controlled by NCPs and by mineral ion availability. Cell processes remain

entrapped within dentin whereas cell bodies remain at the periphery of the pulp.

Adapted from (Vital et al. 2012)

Peritubular dentin result from a passive deposit of serum-derived molecules along the tubule

walls and the crystals form a ring around the tubules lumen (Figure 9). In this type of dentin

no collagen fibrils are detectable, but a thin network of non-collagenous proteins and

phospholipids are visible (M. Goldberg, Molon Noblot, and Septier 1980; Gotliv and Veis

2007; M. Goldberg and Boskey 1996).

21

Figure 9 : Human dentin by scanning electronic microscopy (SEM). A. cutting line is

parallel to dentin tubules, B. cutting line is perpendicular to dentin tubules. PtD:

peritubular dentin, ItD: intertubular dentin. (Own data)

1.2.3.2 Dentin proteins

1.2.3.2.1 Collagens

In the dentin ECM, collagens form a 3D scaffold which is very important in dentinogenesis.

Type I collagen is the major type in dentin matrix collagens (90%), other types of collagen

were identified but at lower levels (1-3%) like types III and V collagens (Michel Goldberg

and Smith 2004; Vital et al. 2012).

Collagen I formed by gathering of two α1 (I) chains and one α2 (I) chain. These chains

entwine to form a triple helix of coiled coil framework (Rest and Garrone 1991). The

22

odontoblasts secrete thin collagen fibril subunits at their apical pole. Lateral fibril subunits

assembly leads to fibrillar growth and then straight integration leads to the collagen

lengthening.

1.2.3.2.2 Noncollagenous proteins (NCPs)

Noncollagenous proteins (NCPs) constitute the remaining 10% of the ECM scaffold and play

an essential role in the regulation of bone and dentin mineralization. NCPs are divided into

phosphorylated and nonphosphorylated NCPs.

1.2.3.2.2.1 Phosphorylated NCPs

SIBLINGs (Small Integrin Binding LIgand N-linked Glycoproteins), are a phosphoprotein

family in which mutations are associated with abnormal phenotypes in the mineralization of

bone and/or dentin (Qin, Baba, and Butler 2004; Vital et al. 2012). This family includes

dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein

(BSP), matrix extracellular phosphorylated glycoprotein (MEPE), and osteopontin (OPN).

All SIBLINGs were identified in dentin and bone ECM, but a high rate of DSPP expression

was shown to be specific to dentin. The SIBLING members carry an arginine– glycine–

aspartate cell adhesion domain (RGD) and a highly conserved acidic serine and aspartate-

rich motif (ASARM) (P. S. Rowe et al. 2000; Fisher and Fedarko 2003). Noteworthy, the

function of ASARM domain in bone and teeth mineralization (apatite crystals nucleator or

inhibitor) is at present debated by the scientific community, in particular its implication in

pathological processes such as inherited rickets (Addison and McKee 2010; David and

Quarles 2010; P. S. N. Rowe 2012). It is of interest that, in addition to binding integrins

SIBLINGs, may also specifically bind and activate several MMPs in the ECM suggesting that

they could be involved in dentin matrix degradation (Fedarko et al. 2004).

1.2.3.2.2.2 Nonphosphorylated NCPs

The second group of NCPs is nonphosphorylated proteins, such as osteonectin (SPARC

protein or BM40) and proteins with gamma-carboxylated glutamates (Gla) residues

(osteocalcin and matrix Gla protein-MGP-). While osteonectin may contribute to the

mineralization process, osteocalcin and MGP have been suggested to regulate HAP crystal

nucleation (Bronckers et al. 1998; Onishi et al. 2005; Kaipatur, Murshed, and McKee 2008).

The small leucine-rich proteoglycans (SLRPs), such as decorin, biglycan, fibromodulin,

lumican, and osteoadherin, have also been identified in predentin and dentin (M. Goldberg,

Septier, and Escaig-Haye 1987; M. Goldberg et al. 2003). They are thought to be involved in

the transport of collagen fibrils through the predentin and in collagen fibrillogenesis (M.

23

Goldberg et al. 2003). Predentin is also rich in dermatan and chondroitin sulphate-containing

(PG). It is of interest that adjacent to the mineralization front, predentin contains a large

quantity of keratan sulphate-containing PG associated with a dramatic decrease in dermatan

and chondroitin sulphate-containing PG. This switch in the proteoglycan type was attributed

to MMP-3, which is closely related to a control of the dentin mineralization process (Hall et

al. 1999).

1.2.3.3 Dentinogenesis

At the early stage of tooth development, the dental mesenchyme originates from the neural

crest-derived mesenchyme migrate to the oral cavity under the oral epithelium and contribute

to the tooth bud formation. During the last mitosis of the proliferate mesenchymal cells the

cell located in contact with the basement membrane become preodontoblasts, whereas the

daughter cells away from the basement membrane form the Hoehl’s layer which constitutes a

reservoir for replacing the damaged odontoblasts. After the differentiation odontoblasts

become polarized and start to secret the extra cellular matrix components which will be the

scaffold for hydroxyapatite (HAP) crystals deposition to form at the end the dentin.

Another classification showed that there are four dentins: Primary dentin, which is formed by

odontoblasts which secret this dentin until the tooth becomes functional. Secondary dentin, is

secreted by odontoblasts immediately after the end of primary dentin secretion (when the

contact of antagonistic cusps is established), and continues throughout life. The major

difference between primary and secondary dentins is morphological; in the secondary dentin

the S-curve of the tubules is more accentuated. Tertiary reactionary dentin, is synthesized by

odontoblasts or, if these cells are destroyed, by the subjacent cells of the Höehl‘s layer, as a

reaction to carious decay, to abrasion or as a response to the release of some components of

dental material fillings. Depending the severity and speed of the carious lesion, the age of the

patient and the progression of the reaction, it appears as a layer of the osteodentin type, or as

a tubular or atubular orthodentin. Tertiary reparative dentin is formed by pulp progenitors,

implicated in the formation of a bone-like or in structure-less mineralization (pulp diffuse

mineralization or pulp stones). These structures are closer to bone (osteodentin) rather than to

dentin (Michel Goldberg et al. 2011).

1.2.4 Enamel

Enamel is the hardest and outer layer of tooth crown that protects the mammalian tooth from

external chemical and physical effects. Enamel properties are associated with its special

structural organization and connection with underlying dentin.(Janet Moradian-Oldak 2012).

24

Mature enamel consists of approximately 4% water and organic material and 96% inorganic

materials (Table 1). Enamel inorganic content is a crystalline calcium phosphate

(hydroxyapatite) which also is found in dentin, cementum, bone, and calcified cartilage

(Antonio Nanci and Cate 2013).

Table 1 : Percentage Wet Weight Composition of Rat Incisor Enamel. From (Antonio

Nanci and Cate 2013)

The principal structural units of enamel are the rods (prisms) and interrod enamel

(interprismatic substance) (Figure 10).

Figure 10 : Scanning electron microscope views of (A) the enamel layer covering

coronal dentin, (B) the complex distribution of enamel rods across the layer, (C and D)

25

and perspectives of the rod-interrod relationship when rods are exposed longitudinally

(C) or in cross section (D). Interrod enamel surrounds each rod. DEJ: Dentinoenamel

junction; IR: interrod; R,rod. Adapted from (Antonio Nanci and Cate 2013)

1.2.4.1 Enamel proteins

Enamel proteins are synthesized by ameloblasts. During tooth development, the ameloblasts

control the synthesis and secretion of the organic extracellular matrix (ECM) and then the

biomineralization of this ECM. Enamel proteins are hydrophobic proteins known such as

amelogenins and nonamelogenin proteins including ameloblastin, enamelin, tuftelin, tuft

proteins, sulfated proteins and enamel proteases such as enamelysin (MMP-20) and KLK-4.

1.2.4.1.1 Amelogenin

Amelogenin gene exists only on the X chromosome in rodents (Snead et al. 1983; Chapman et

al. 1991), while it exists on both X and Y chromosomes In human and cow (Lau et al. 1989).

Amelogenin constitutes more than 90% of the enamel protein content. It is secreted as a

variety of isoforms because of alternative splicing of the amelogenin gene and processing of

the parent molecules (C. W. Gibson et al. 1991; Lau et al. 1992), the major isoform is about

25 kDa. Amelogenin has a bipolar nature: it contains highly hydrophobic domains and

hydrophilic N- and C-terminal sequences and this bipolar nature allows amelogenin

monomers by self-assembly to form supramolecular resulting in the formation of nanospheres

which regulate crystal spacing (Fincham et al. 1994; Fincham and Simmer 1997). The N-

terminal A-domain is involved in the formation of nanospheres, whereas the carboxy-

terminal B-domain prevents their fusion to larger assemblies (J. Moradian-Oldak et al. 2000).

Amelogenin has signaling activities (Carolyn W. Gibson 2008; Veis 2003), especially the

small isoform; leucine-rich amelogenin peptide (LRAP) (Warotayanont et al. 2008). Because

of its potential to promote cell differentiation and its interaction with bone cells, it has been

used in periodontal regenerative therapies.

Amelogenin is not essential for the initiation of mineralization, but is essential for the

elongation of enamel crystals and the achievement of proper enamel formation, because in

spite of its absence in KO mice, a thin layer of mineralized enamel is formed.

1.2.4.1.2 Ameloblastin

Ameloblastin constitutes about 5% of enamel protein. Its expression significantly decrees

during enamel maturation. The isolation of this protein is so difficult for several reasons, the

26

limitations in expression and hydrolysis by enamel proteinase MMP-20 as soon as secreted

(Iwata et al. 2007; Yasuo Yamakoshi, Hu, Zhang, et al. 2006). In the ameloblastin KO mice,

ameloblasts detach from the surface of the developing teeth, suggesting a potential role for

ameloblastin in ameloblasts adhesion to the forming enamel (Fukumoto et al. 2004).

1.2.4.1.3 Enamelin

Enamelin is the largest enamel protein and constitutes about (3–5%) of enamel proteins. It is

a phosphorylated, glycosylated protein and is rapidly cleaved following its secretion. The

intact protein is only observed at the mineralization front, so it proposed to be implicated in

crystal elongation (C. C. Hu et al. 1997; C. C. Hu et al. 2000).

Enam gene mutations cause an autosomal dominant forms of amelogenesis imperfecta AI

(Hart et al. 2003) and no true enamel layer is formed in the Enam KO mice(J. C.-C. Hu et al.

2008).

Recently, it was reported that a large increase or decrease in enamelin expression impairs the

production of enamel crystals and the prism structure (J. C.-C. Hu et al. 2014).

Enamelin and ameloblastin appear to have similar roles like crystallite initiation and

elongation, whereas amelogenin appears to form a framework to allow the continued

elongation of the already initiated crystallites (John D. Bartlett 2013).

1.2.4.1.4 Tuftelin

Tuftelin is expressed early at the bud stage of tooth development (several days befor the onset

of mineralisation) so it is suggested to play a nucleator role during crystals formation. Its

expression is also detected in several organs kidney, lung, liver, and testis (Zeichner-David

et al. 1997; MacDougall et al. 1998).

1.2.4.1.5 Sulfated enamel proteins

Sulfated enamel proteins constitute an acidic nature family of proteins with unknown roles.

They are difficult to be detected because of their presence in a small amount (C. E. Smith et

al. 1995).

1.2.4.1.6 Amelotin

Amelotin is a glycoprotein recently discovered, its role is not yet clear (Iwasaki et al. 2005).

It is expressed during the secretory stage of enamel development (Gao et al. 2010).

Alternatively spliced variants lead to several isoform of amelotin.

27

1.2.4.1.7 Biglycan

Biglycan expression is in the dentin and the enamel (Septier et al. 2001). It is expressed by

ameloblasts during tooth development, (M. Goldberg, Septier, Rapoport, et al. 2002), where

it acts as an amelogenin expression repressor (M. Goldberg, Septier, Rapoport, et al. 2002;

M. Goldberg et al. 2005b).

1.2.4.2 Enamel proteinases

Enamel proteinases are so important for the digestion of enamel proteins and enamel

maturation. It was found that some of these proteinases have an ameloblast differentiation-

dependent expression (Lu et al. 2008).

1.2.4.2.1 Matrix metalloproteinase 20 (MMP-20)

Enamelysin (MMP-20) is expressed by ameloblasts and odontoblasts (J. D. Bartlett et al.

1996; Fukae and Tanabe 1998), it is expressed from the beginning of secretion stage through

the beginning of maturation stage of enamel and cleaves amelogenin, enamelin, and

ameloblastin into stable intermediate products (Lacruz et al. 2011). In vitro studies showed

that Mmp-20 stimulates the formation of nanorod structures formed by co-assembly of the

parent amelogenin with its proteolytic products (X. Yang et al. 2011). Such assembly

alteration was proposed to be related with the elongated growth of apatite crystals. It has been

proposed that Mmp-20 activity produces protein intermediate products that will stimulate

phase transformation of amorphous calcium phosphate nanoparticles into mineralized

hydroxyapatite (Kwak et al. 2009).

1.2.4.2.2 Kallikrein-4 (KLK4)

Klk-4 is expressed from the end of secretory stage and throughout the maturation stage of

enamel (Lacruz et al. 2011). Its function is to digest the intermediate products of amelogenin,

enamelin and ameloblastin resulting from the MMP-20 action and facilitates enamel proteins

removal which is necessary for enamel maturation and hardening (O. Ryu et al. 2002).

KLK-4 digests the 32-kDa enamelin fragment which is resistant to Mmp-20 action, (Yasuo

Yamakoshi, Hu, Fukae, et al. 2006) and its activity is not affected like MMP-20 by the

presence of apatite crystals in vitro (Z. Sun et al. 2010).

1.2.4.2.3 Other proteinases

1.2.4.2.3.1 Caldecrin (Ctrc)

Caldecrin Ctrc expression pattern in enamel is similar to Klk4, but lower, and it is

predominantly expressed in the maturation stage of amelogenesis (Lacruz et al. 2011).

28

1.2.4.2.3.2 MMP-2

It has been demonstrated that recombinant MMP-2 cleave amelogenin into several fragments

in vitro (Caron et al. 2001). MMP-2 also degraded most forms of amelogenin, suggesting that

MMP-2 can participate, with MMP-20, to achieve complete amelogenin processing (Bourd-

Boittin et al. 2005).

1.2.4.2.3.3 MMP-9

Recently, it was proposed that MMP-9 involved in enamel formation and controlling the

processing of amelogenin (Feng et al. 2012)

1.2.4.3 Enamel formation

1.2.4.3.1 Pre-secretory stage

At this stage, ameloblasts start expressing very small amounts of enamel proteins even before

the basement membrane break up and send cytoplasmic projections through the gaps directly

after basement membrane disintegrate. With the disappearance of the basement membrane,

dentin starts to mineralize and the apical surfaces of ameloblasts connect with the superficial

collagen fibrils of the mantle dentin (Meckel, Griebstein, and Neal 1965; Cevc et al. 1980)

(Figure 12).

1.2.4.3.2 Secretory Stage

At the beginning, ameloblasts secrete enamel proteins on top of and around existing dentin

crystals initially and then around enamel crystals and into the space of disappeared basement

membrane (Figure 11.A). With the continued secretion of enamel matrix, ameloblasts move

back to create the necessary space for continuous deposition of enamel end this moment we

can distinguish the initial enamel layer which is aprismatic (not separated into rod and

interrod enamel)

Secretory ameloblasts develop a novel cell extension called Tomes’ process at their apical

(secretory) ends. This extension which has secretory and nonsecretory regions provides the

architectural basis for organizing enamel crystals into rod and interrod enamel, (Meckel,

Griebstein, and Neal 1965; Cevc et al. 1980).

Secretory ameloblasts secrete enamel proteins which concentrate along the ameloblast

secretory membrane and form a mineralization front (there is no pre-enamel like the

predentin in dentin or osteoid in bone). The mineralization front retreats with the Tomes’

process as the enamel crystals grow in length (4µm/day) (Risnes 1986), and the ameloblasts

29

continue their secretion of enamel proteins (Figure 12). During this stage enamel crystals

grow primarily in length and the enamel layer thickens.

Figure 11 : Semi-thin (0.5 µm) sections from glutaraldehyde-fixed, decalcified, and

plastic embedded mandibular incisors of wild-type mice stained with toluidine blue to

illustrate the appearance of enamel and enamel organ cells at mid-secretory stage (A)

and near-mid-maturation stage (B) of enamel development. Abbreviations: E, enamel;

Am, ameloblast; Si, stratum intermedium; pd, predentin; D, dentin; ae, apical end; be,

basal end; bv, blood vessel; as, artifact space; b, bone; c, cementum. Adapted from (J.

D. Bartlett and Smith 2013)

1.2.4.3.3 Maturation Stage

At the end of secretory stage, enamel layer has its final thickness and ameloblasts reduce their

secretion of enamel proteins (Figure 11.B), and start the secretion of KLK-4 which finishes

the degradation of the organic matrix. The degradation and removal of growth-inhibiting

enamel proteins terminate the growth of enamel crystallites in length, and accelerate their

growth in width and thickness by the ion deposition on the thin crystals sides until they press

against one another (C. E. Smith 1998). This process is necessary to have a harde and mature

30

enamel layer, and is directed by modulating ameloblasts that cycle through smooth and

ruffle-ended phases.

During maturation stage a basal lamina is secreted at the base of the ameloblasts (Figure

12). Recently amelotin ( AMTN ) has been identified as one of the components of this basal

lamina (Iwasaki et al. 2005; Moffatt et al. 2006).

Figure 12 : Schema present incisor enamel and denin formation. p-Am: pre-ameloblast;

pOd: pre-odontoblast; s-Am: secreting Ameloblasts; od: odontoblasts; pos-Am: post-

secretory ameloblasts; pD: predentin; D: dentin; pm-E: premature enamel; E: enamel.

Adapted from (Khaddam et al. 2014)

1.3 Matrix MetalloProteinases MMPs

MMPs is subdivided into soluble and membrane-type MMPs (MT-MMPs). The soluble

MMPs are expressed as pro-enzymes that will be activated in the extracellular environment.

MT-MMPs are intracellularly activated and identified as activators of soluble MMPs and

were shown to be able to degrade extracellular matrix proteins ECM (Hamacher, Matern, and

Roeb 2004).

31

In addition to the inhibition by endogenous inhibitors (tissue inhibitor of MMPs TIMP) or to

the proteolytic activation of pro-MMPs, MMPs are regulated by cytokines or growth factors

transcriptionally (Tsuruda, Costello-Boerrigter, and Burnett 2004)

MMPs are implicated in inflammation by regulating the availability and the activity of

cytokines, chemokines, and growth factors, as well as integrity of tissue barriers. MMPs are

also involved in tumors (Nissinen and Kähäri 2014).

1.3.1 MMPs and teeth

1.3.1.1 In physiological processes (development)

Several MMPs have been detected in developing tooth tissues (Michel Goldberg et al. 2003).

They play a central role in the disruption of basement membrane. MMPs are also implicated

in the functional regulation of growth factors and their receptors, cytokines and chemokines,

adhesion receptors and cell surface proteoglycans, and a variety of enzymes (H. Li et al.

2002). MMPs participate in the remodeling of the ECM during tooth development to

facilitate the migration of cells and the mesenchymal condensation (Chin and Werb 1997)

and participate in the regulation of the mineralization process of dental hard tissues by

cleaving the matrix proteins of the dentin and the enamel matrix (Simmer and Hu 2002;

Fanchon et al. 2004).

MMP-1, -2, -3, -9 and MT1-MMP have been detected during tooth development, indicating

that these MMPs have roles in tooth morphogenesis and eruption (Chin and Werb 1997;

Sahlberg et al. 1992b; Caron, Xue, and Bartlett 1998; Randall and Hall 2002; Yoshiba et al.

2003).

1.3.1.2 In pathological processes

1.3.1.2.1 Periodontitis

High MMPs levels were detected in the periodontitis and apical periodontitis leading to

accelerated matrix degradation, (de Paula e Silva et al. 2009; Paula-Silva, da Silva, and

Kapila 2010). Collagenases (MMP-1, MMP-8, and MMP-13) and gelatinases (MMP-2 and

MMP-9) are implicated in the digestion of collagen in the bone and periodontal ligament

(Andonovska, Dimova, and Panov 2008; Corotti et al. 2009).

1.3.1.2.2 In the caries process

We developed this point (Figure 13) in Chaussain, Boukpessi, Khaddam et al, 2013 (end of

introduction).

32

Figure 13 : Schematic representation of MMP activity during the dentin carious

process. Cariogenic bacteria present in the caries cavity release acids such as lactic acid

that reduce the local pH. The resulting acidic environment demineralizes the dentin

matrix and induces the activation of host MMPs derived from dentin or saliva (which

bathes the caries cavity). Once the local pH is neutralized by salivary buffer systems,

activated MMPs degrade the demineralized dentin matrix. Adapted from (Chaussain et

al. 2013)

1.4 EMMPRIN (Basigin,CD147)

1.4.1 Historic

Extra cellular matrix metalloproteinase inducer EMMPRIN (CD147), a member of the

immunoglobulin superfamily, was described for the first time on the surface of solid

tumor cells as an inducer of a various (MMPs in adjacent fibroblasts (Biswas 1982).

Based on these latter properties it was named extracellular matrix metalloproteinase

inducer EMMPRIN (Biswas et al. 1995). Previously EMMPRIN had different names

including tumor cell-derived collagenase stimulatory factor (TCSF), Basigin, CD147,

gp42, HT7, neurothelin, 5A11, OX-47 and M6 (T. Muramatsu and Miyauchi 2003).

1.4.2 Structure

1.4.2.1 Transmembrane form

EMMPRIN (Basigin) has four isoforms (basigin-1 to -4), caused by alternative transcription

initiation and variation in splicing (Figure 14)(Belton et al. 2008) and the major isoform is

basigin-2. EMMPRIN is highly glycosylated, Its protein portion is 27 kDa, and its

33

glycosylated form is 43 to 66 kDa (Miyauchi et al. 1990) and the nonglycosylated form has

the ability to induce MMP expression in fibroblasts as the glycosylated form(Belton et al.

2008)

Figure 14: Basigin isoforms. Characteristic features of isoforms are mentioned within

blanket. Carbohydrates are shown by light grey color. Adapted from (Takashi

Muramatsu 2012)

EMMPRIN is largely composed of three domains, extracellular immunoglobulin domain, a

transmembrane domain and a cytoplasmic domain.

The extracellular domain has two immunoglobulin domains (a N-terminally located D1

domain and a more C-terminally located D2 domain) (Figure 15) and three potential

Asparagine (Asn)-glycosylation sites; one in D1 domain and two in D2 domain (Miyauchi et

al. 1990; Miyauchi, Masuzawa, and Muramatsu 1991).

The transmembrane domain has glutamic acid in its center, and is completely conserved

between human, mouse and chicken (Miyauchi, Masuzawa, and Muramatsu 1991), this

domain is important for interactions with other proteins in the same membrane.

34

Figure 15: Scheme of EMMPRIN structure. EMMPRIN contains an extracellular

domain composed of two Ig loops with three Asn-linked oligosaccharides and short

single transmembrane domain (TM) and a cytoplasmic domain (Cyt). The first Ig

domain is required for counter-receptor activity, involved in MMP induction. Adapted

from (Gabison et al. 2009).

1.4.2.2 Soluble form

The soluble form of CD147 has been detected in conditioned media as:

full-length protein (Taylor et al. 2002)

or as part of shed microvesicles (Sidhu et al. 2004)

as well as in forms lacking the transmembrane and cytoplasmic domain derived from

MMP mediated cleavage of CD147 from the cell surface (Haug et al. 2004; Y. Tang

et al. 2006; Egawa et al. 2006)

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1.4.3 Phenotypes of EMMPRIN knock out (KO) mice

EMMPRIN KO mice have a low reproduction level which is at a much lower frequency than

that expected by Mendelian segregation, KO embryos develop normally during blastocyst

stage but at the time of implantation, about 75% of the null embryos are lost (Igakura et al.

1998) and half of the surviving mice had interstitial pneumonia and died within 4 weeks after

birth (Igakura et al. 1998). EMMPRIN KO mice have a defect in the capability of

implantation of the uterus (female), arrested spermatogenesis (male) (Igakura et al. 1998;

Kuno et al. 1998), abnormal behavior (Naruhashi et al. 1997), deficits in vision (Hori et al.

2000) and a decreased response to odor (Igakura et al. 1996).

1.4.4 EMMPRIN interactions

Three possible EMMPRIN interactions were descriped (Figure 16):

- Homophilic cis interaction between EMMPRIN molecules within the plasma

membrane of the same tumor cell (Yoshida et al. 2000).

- Homophilic trans interaction between EMMPRIN molecules on tumoral cells.(J. Sun

and Hemler 2001)

- Heterophilic interactions between EMMPRIN molecule on a tumor cell and a putative

EMMPRIN receptor on a fibroblast.

Figure 16: Possible EMMPRIN-mediated interactions stimulating MMP production.

(A) Homophilic cis interaction between EMMPRIN molecules within the plasma

membrane of a tumor cell. (B) Homophilic trans interaction between EMMPRIN

molecules on apposing tumor cells. (C) Heterophilic interactions between EMMPRIN

36

on a tumor cell and a putative EMMPRIN receptor on a fibroblast. Adapted from

(Toole 2003)

1.4.4.1 EMMPRIN Interactions with its binding partners within cell membrane

1.4.4.1.1 Monocarboxylic acid transporters (MCTs(

EMMPRIN has multiple binding partners, one of them, a family of monocarboxylic acid

transporters (MCTs) (Kirk et al. 2000; Halestrap 2012) which transport monocarboxylic acids

such as lactate, pyruvate and ketone bodies into and from the cells. Among the four MCTs

(MCT1, MCT2, MCT3 and MCT4), EMMPIN binds to MCT1, MCT3 and MCT4 in the

same membrane, and is essential for their transfer to the cell surface. An EMMPRIN dimer

binds two MCT1 (Wilson, Meredith, and Halestrap 2002).

1.4.4.1.2 Integrins

It was shown that EMMPRIN associates with integrin α3 β1 and α6 β1 in the same

membrane (Berditchevski et al. 1997), for example in extraembryonic membrane apposition

in D. melanogaster (Reed et al. 2004) and in the visual system of D. melanogaster (Curtin,

Meinertzhagen, and Wyman 2005).

1.4.4.1.3 Caveolin-1

Caveolin is a family of proteins form the major constituents of caveolae, within the plasma

membranes of most cells that mediate the transcytosis of macromolecules in a clathrin-

independent manner (Williams and Lisanti 2005) and comprised of three isoforms (caveolins

1, 2 and 3), only one of them caveolin-1 has been shown to associate with EMMPRIN. The

association starts within the Golgi apparatus, where caveolin-1 binds to and guard the lower

glycosylated forms of EMMPRIN to the plasma membrane, thus prevents the formation of,

highly glycosylated species of EMMPRIN by the self-aggregating, which is responsible for

MMP production (W. Tang, Chang, and Hemler 2004).

Caveolin-1 serves as a negative regulator of EMMPRIN; by the direct association with the

second Ig domain of EMMPRIN which decrease EMMPRIN clustering and resulted in

decreased MMP production (W. Tang and Hemler 2004).

Recently, an opposite effect of caveolin-1 is demonstrated. The increased caveolin-1

expression results in an increased proportion of highly glycosylated EMMPRIN relative to

the lower glycosylated form and increased production of MMP-11 and higher invasiveness.

In the same study down-regulation of caveolin-1 resulted in a decrease in highly glycosylated

37

EMMPRIN (Jia et al. 2006). Regardless of the outcome of these studies, the expression of

EMMPRIN glycosylation forms is functionally linked with caveolin-1 expression.

1.4.4.2 EMMPRIN interactions with external molecules

1.4.4.2.1 Cyclophilin

Cyclophilin A is secreted from cells exhibit high level chemotactic activity to leukocytes and

is involved in the inflammation, so it is the target protein of an immunosuppressive drug,

cyclosporine A. Several studies confirmed that EMMPRIN is the receptor for cyclophilin A

(V. Yurchenko et al. 2010; Vyacheslav Yurchenko et al. 2002), and for cyclophilin B (V.

Yurchenko et al. 2010). The affinity between cyclophilin A and the extracellular region of

EMMPRIN is weak, but it is strong with the transmembrane region (Schlegel et al. 2009).

1.4.4.2.2 EMMPRIN

Recently it has shown that nonglycosylated EMMPRIN ectodomains form dimer, and then

interact with EMMPRIN on target cells (Belton et al. 2008). During internalization,

EMMPRIN associates with another form of EMMPRIN (basigin-3 (Belton et al. 2008).

1.4.4.2.3 Platelet glycoprotein VI (GPVI)

Recently, platelet glycoprotein VI (GPVI) has been identified as a novel receptor for

EMMPRIN and can mediate platelet rolling via (Seizer et al. 2009).

1.4.5 EMMPRIN functions

1.4.5.1 In physiological processes

1.4.5.1.1 Tissue repair/remodeling

The balance between MMP-induced stromal remodeling/restoration and stromal destruction

is so delicate. EMMPRIN has been proposed as a mediator for this balance directly via a

feedback mechanism that links the affected epithelial cells with neighboring fibroblasts

(Gabison, Hoang-Xuan, et al. 2005).

In corneal ulcerations the protective epithelial barrier of the eye is damaged, leaving eye open

to infection by bacteria, viruses and fungi and, if left untreated, they can result in blindness.

EMMPRIN has been detected in both healthy and ulcerated corneas but was found at greater

levels within ulcerated specially at the areas of greater MMP expression (Gabison, Mourah,

et al. 2005).

38

Within the cardiovascular system, the balance is also delicate. MMP expression is critical for

the prevention of hypertension and, at the same time, implicated in the progression of

congestive heart failure (CHF) (Spinale et al. 2000; Ergul et al. 2004).

1.4.5.1.2 Chaperone functions

It was shown that In MCT-transfected cells, the MCT1 and MCT4 expressed protein

accumulated in a perinuclear compartment, and it was found that co-transfection with CD147

enabled plasma membrane expression of active MCT1 or MCT4. Showing that EMMPRIN

facilates proper expression of MCT1 and MCT4 at the cell surface and have a chaperone

function (Kirk et al. 2000).

1.4.5.1.3 Implantation

Since MMPs are required in implantation (Alexander et al. 1996; Werb 1997), defective

implantation result from mis-regulation of MMP production due to lack of EMMPRIN

stimulation in EMMPRIN KO mice.

1.4.5.1.4 Spermatogenesis

EMMPRIN is strongly expressed in spermatocytes (Igakura et al. 1998) and its absence in

EMMPRIN KO mice, leads to arrest spermatogenesis at the stage of differentiation of

primary spermatocytes into secondary spermatocytes at the metaphase of the first meiosis

(Igakura et al. 1998).

1.4.5.1.5 Retinal development and maintenance

EMMPRIN mediates MCTs transport to the plasma membrane so MCT1, MCT3 and MCT4

were found to be deficient at the plasma membrane of the retinal pigment epithelia, which

leads abnormal photoreceptor function and blindness (Hori et al. 2000; Philp et al. 2003).

1.4.5.1.6 Cell interactions

EMMPRIN mediates the adhesive cell interactions, like in the embryonic retinal cell

aggregation and influences glial cell maturation (Fadool and Linser 1993).

1.4.5.1.7 Hematopoetic cell activation and erythrocytes circulation

It was found that EMMPRIN plays a role in hematopoietic cell activation as during dendritic

cell differentiation (Cho et al. 2001; Spring et al. 1997). Furthermore, It has been shown that

EMMPRIN is expressed in erythrocyte lineage cells, including mature erythrocytes and

blocking EMMPRIN by the injection of a monoclonal antibody in the mice causes selective

trapping of erythrocytes in the spleen (Coste et al. 2001).

39

1.4.5.1.8 Other

EMMPRIN is implicated in other several physiological processes like neural network

formation and development (Schlosshauer 1991; Fadool and Linser 1993), restriction of

synaptic bouton size (Besse et al. 2007), calcium transport (J. L. Jiang et al. 2001), neutrophil

chemotaxis (Vyacheslav Yurchenko et al. 2002), and blood –brain barrier development

(Schlosshauer 1993).

1.4.5.2 In pathological processes

1.4.5.2.1 Cancer

High levels of EMMPRIN were reported in numerous malignant tumors including bladder,

skin, lung and breast carcinoma, and lymphoma (Polette et al. 1997; Bordador et al. 2000;

Thorns, Feller, and Merz 2002), and were also associated with poor prognosis (Kanekura,

Chen, and Kanzaki 2002; Davidson, Givant-Horwitz, et al. 2003; Davidson, Goldberg, et al.

2003; Ishibashi et al. 2004).

EMMPRIN induces several malignant properties associated with cancer. These include:

1.4.5.2.1.1 MMPs

Tumorigenic cells expressing EMMPRIN induce MMP expression by neighboring stromal

cells (Figure 17) (Biswas 1982; Kataoka et al. 1993; Biswas et al. 1995) and regulates MMP

production at the transcription level by a mitogen activated protein kinase(MAPK) p38

pathway (Lim et al. 1998; Lai et al. 2003). Both recombinant EMMPRIN and tumoral

EMMPRIN have been shown to induce the expression of collagenase I (MMP-1), gelatinase

A (MMP-2), stromelysin-1 (MMP-3), and membrane type 1- and type 2-MMPs (MT1- and

MT2-MMP) by fibroblasts (Cao, Xiang, and Li 2009; J. Sun and Hemler 2001; R. Li et al.

2001; Sameshima et al. 2000).

In situ hybridization analyses of both tumor and peri-tumoral fibroblasts in different organs

like breast, colon, lung, skin and head/neck tumors showed that EMMPRIN expression is

primarily tumor associated, while MMP expression is fibroblast associated (Pyke et al. 1992;

Majmudar et al. 1994; Noël et al. 1994; Heppner et al. 1996; Polette et al. 1997).

EMMPRIN can induce EMMPRIN and MMP expression in far stromal cells by its soluble

form which result from proteolytic cleavage of the carboxy terminus, and is thought to help

metastasis to distant sites (Y. Tang et al. 2004).

40

Recently, another mechanism for MMP stimulation in distant cells was described. It was

found that EMMPRIN expressed by malignant testicular cells by membrane vesicles (MVs)

secreted from these cells, can exert its MMP inducing effect on distant cells within the tumor

microenvironment to promote tumor invasion (Milia-Argeiti et al. 2014).

These properties make EMMPRIN a good target in cancer therapy. It has been shown that

antibodies to EMMPRIN can decrease MMP expression leading to an inhibition of tumor cell

invasion (Bordador et al. 2000; J. Sun and Hemler 2001; Kanekura, Chen, and Kanzaki

2002).

Figure 17 : Tumor-cell induced activation of adjacent fibroblasts by homophilic

EMMPRIN signaling. Adapted from (Joghetaei et al. 2013)

1.4.5.2.1.2 VEGF

VEGF works as a major regulator of the angiogenic process in different circumstances,

including tumor formation. EMMPRIN, in addition to increasing tumor invasion through

MMP induction, it induces angiogenesis by the up-regulation VEGF expression (Y. Tang et

al. 2005) as well as the stimulation of cell survival signaling, including Akt, Erk and FAK,

through the increased production of the pericellular polysaccharide hyaluronan (Toole and

41

Slomiany 2008). EMMPRIN regulates VEGF production in tumor and fibroblast cells via the

PI3K-Akt pathway (Y. Tang et al. 2006).

1.4.5.2.1.3 HIF-1α and MCT

The increase of tumor size makes the tumor microenvironment suffering from hypoxia and

induce the hypoxia inducible factor, HIF-1 α , a transcription factor which has been shown to

induce MCT-4 gene expression in cells (Moeller, Dumitrescu, and Refetoff 2005; Moeller et

al. 2006). Up-regulation of MCTs in tumor cells is necessary for tumor survival and increase

lactic acid concentration in the tumoral extracelluar microenvironment. This excess lactic

acid inhibits peritumoral cytotoxic T cell function, and permitting continued uncontroled

growth of the tumor (Fischer et al. 2007). MCT up-regulation is coordinated with EMMPRIN

up-regulation which induces MMP production by peritumoral fibroblasts resulting in the

extracellular matrix degradation and a favorable environment for tumor metastasis.

1.4.5.2.2 Rheumatoid arthritis

In rheumatoid arthritis (RA), Cyclophilin A (Cyp-A) up-regulates the expression of MMP-9

via the EMMPRIN signaling pathway through direct binding to EMMPRIN (Y. Yang et al.

2008). And recently, it was found that EMMPRIN induces up-regulation of HIF-1α and

VEGF in RA fibroblast-like synoviocytes, which promotes angiogenesis, and leads to the

persistence of synovitis (C. Wang et al. 2012).

1.4.5.2.3 Ischemic disease

The oxygen level decreases in the heart during myocardial infarction and in the brain during

stroke. Because of hypoxia and ischemia cells become dependent upon glycolysis for energy

metabolism, for continued cell viability the EMMPRIN associated lactate transporters MCT-

1 and MCT-4 will be necessary (Kirk et al. 2000). High levels of MCT and EMMPRIN

expression are detected under ischemic conditions in cardiac and neuronal cells (F. Zhang et

al. 2005; Han et al. 2006), and it has been reported that EMMPRIN/Cyclofilin A association

protects neurons from ischemia and hypoxia (Boulos et al. 2007).

1.4.5.2.4 Graft-versus-host disease

Using monoclonal antibody to EMMPRIN as a treatment for patients exhibiting acute graft-

versus-host disease, shows promising efficacy, this effect due to suppression of leukocyte

activation (Deeg et al. 2001)

1.4.5.2.5 Other diseases

Role of EMMPRIN is reported in other processes like atherosclerosis (L. Liang, Major, and

Bocan 2002), heart failure (Y. Y. Li, McTiernan, and Feldman 2000; Spinale et al. 2000),

42

lung injury (Foda et al. 2001), viral infection (Pushkarsky et al. 2001), Alzheimer’s disease

(Zhou et al. 2005), chronic liver disease (Shackel et al. 2002) and in lymphocyte migration

and activation (Koch et al. 1999; Renno et al. 2002; X. Zhang et al. 2002) .

43

1.4.6 EMMPRIN and tooth

1.4.6.1 In physiological processes

During tooth development, in cap stage, EMMPRIN expression was detected in the cell

membranes of the inner enamel epithelium, stratum intermedium cells of the enamel organ

and the dental papilla cells underlying the inner epithelium (Figure 18.a) (Kumamoto and

Ooya 2006; Schwab et al. 2007; Xie et al. 2010; S.-Y. Yang et al. 2012). In bell stage, it was

detected in ameloblasts, stratum intermedium, and in odontoblasts (Figure 18.b) (Schwab et

al. 2007; Xie et al. 2010; S.-Y. Yang et al. 2012).

Figure 18 : Immunoreactivity (IR) for EMMPRIN. a Cells of the inner enamel

epithelium (cap stage of the enamel organ) show intense EMMPRIN IR (Alexa-

coupled). b Ameloblasts as well as odontoblasts (bell stage of the enamel organ) exhibit

strong EMMPRIN IR. Note the IR in the borderline between ameloblasts and the

stratum intermedium. Mesenchymal cells of the dental papilla are only weakly

immunoreactive. Abbreviations: A ameloblast; DL dental lamina; EEE external enamel

epithelium; IEE internal enamel epithelium; EO enamel organ; Od odontoblast; SI

stratum intermedium; SR stellate reticulum. Adapted from (Schwab et al. 2007)

EMMPRIN variability during tooth development was investigated, and it was found that

EMMPRIN mRNA expression was higher in E13.0 mouse mandible than that in E11.0

(Figure 19.a). and was higher in P1 mouse tooth germ than that in E14.0 (Figure 19.b) (Xie

et al. 2010).

44

Figure 19 : Transcription level of EMMPRIN in different stages of tooth development. a

EMMPRIN mRNA was higher in E13.0 mandible than that in E11.0. b The expression

of EMMPRIN mRNA was higher in P1 tooth germ than that in E14.0. Adapted from

(Xie et al. 2010)

At the root formation stage of tooth development, EMMPRIN was expressed strongly in the

follicular cells overlaying the occlusal region of the rat molar germs. But, the expression was

not region-specific and was weak in the follicular tissues in molar germs at the cap stage. So

it was suggested that EMMPRIN play role in dental hard tissue maturation and the formation

of an eruption pathway (S.-Y. Yang et al. 2012).

The differentiation-dependent co-expression of EMMPRIN with MMPs in the odontoblasts

and enamel organ indicates that EMMPRIN plays role in proteolytic enzymes induction in

the rat tooth germ (Schwab et al. 2007). And it was found that EMMPRIN colocalizes with

caveolin-1 in cell membranes of ameloblasts and in inner enamel epithelial cells (Schwab et

al. 2007).

EMMPRIN functional role in tooth germ development was investigated, by an EMMPRIN

siRNA interference approach. Significant increase in MT1-MMP mRNA expression and a

reduction in MMP-2, MMP-3, MMP-9, MMP-13 and MT2-MMP mRNA expression were

observed in the mouse mandibles following EMMPRIN abrogation. These results indicate

that EMMPRIN could be involved in the early stage of tooth germ development and

morphogenesis (Figure 20), possibly by regulating the MMP expression (Xie et al. 2010).

45

Figure 20 : Examination the role of EMMRIN in early tooth germ development using

EMMPRIN siRNA in the cultured mandible at E11.0. a After being cultured for 6 days,

the tooth germ was found to have developed into the cap stage in mandibles cultured

with scramble siRNA. b Dental epithelial bud was observed in the mandible treated

with EMMPRIN siRNA after 6 days of culture. c A cap-like mature enamel organ was

observed in the mandibles with scrambled siRNA supplement at 8-day culture. d

EMMPRIN siRNAtreated mandible explants also showed a bud-like tooth germ at 8-

day culture. EMMPRIN siRNA had a specific effect on the morphogenesis of tooth

germ. DE dental epithelium, DM dental mesenchyme, DP dental papilla, EO enamel

organ, OE oral epithelium, PEK primary enamel knot. Adapted from (Xie et al. 2010)

1.4.6.2 In pathological processes

Several reports have pointed to the relation between periodontitis and EMMPRIN (Dong et

al. 2009; Xiang et al. 2009; L. Liu et al. 2010; Feldman et al. 2011; D. Yang et al. 2013; J.

Wang et al. 2014).

Elevated levels of EMMPRIN have been related to the progression of periodontal disease

(Dong et al. 2009). EMMPRIN expression level increased from day 3 to day 7 and then

gradually decreased from day 11 to day 21 (L. Liu et al. 2010; D. Yang et al. 2013).

During periodontitis development EMMPRIN was detected in the interdental gingiva, the

gingival epithelium (Figure 21) and adjacent fibroblasts and in the inter-radicular bone. its

46

inflammation-dependent expression was associated with collagen breakdown and alveolar

bone loss (L. Liu et al. 2010).

Figure 21 : Temporal expression and localization of EMMPRIN in the gingival

epithelium during ligature-induced periodontitis in the first mandibular molar of rats.

(A) On day 0 (health), the immunoreactivity was strong in the basal cells, with a

decrease toward the upper layers in the attached gingival epithelium (star in a1). (B) On

day 7, immunoreactivity was greatly enhanced in the attached gingiva (star in b1). (C)

On day 15, immunoreactivity was similar to that seen in the healthy state in the

attached gingival epithelium (star in c1). Adapted from (L. Liu et al. 2010)

EMMPRIN relation with other proteins during periodontitis was studied, it was found that

EMMPRIN is associated with MMP-13 (higher expression level in day 3) but not with MMP-

8 (higher MMP-8 expression in day 3) (D. Yang et al. 2013), and it was found that the

increased active MMP-1 and proMMP-1 production in the chronic human periodontitis may

be associated with elevated HG-EMMPRIN levels (J. Wang et al. 2014).

Soluble forms of EMMPRIN were shown to be present in gingival crevicular fluid (GCF) of

patients with different periodontal diseases for the first time by Emingil et al. These authors

showed that elevated EMMPRIN levels in gingival crevicular fluid were related to the

47

enhanced severity of periodontal inflammation, indicating that EMMPRIN may participate in

the regulation of periodontal disease progression (Emingil et al. 2006).

High EMMPRIN level was detected in human ameloblastomas (L.-J. Jiang et al. 2008;

Kumamoto and Ooya 2006; Er et al. 2001), oral squamous cell carcinoma (Bordador et al.

2000), and odontogenic cysts (L.-J. Jiang et al. 2008; Ali 2008). EMMPRIN expression was

significantly higher in ameloblastomas than in odontogenic cysts, and microvessel density

was positively associated with EMMPRIN expression to some extent (L.-J. Jiang et al. 2008).

No significant difference in EMMPRIN expression was found among tumor types or

subtypes (Kumamoto and Ooya 2006). EMMPRIN expression variability in various types of

odontogenic cysts was studied and it was found that EMMPRIN expression was significantly

higher in the epithelial lining of odontogenic keratocysts than in the dentigerous and

periapical cysts (Ali 2008).

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2 MMPs and dentin matrix degradation

Chaussain, Catherine, Tchilalo Boukpessi, Mayssam Khaddam, Leo Tjaderhane, Anne

George, and Suzanne Menashi. 2013. “Dentin Matrix Degradation by Host Matrix

Metalloproteinases: Inhibition and Clinical Perspectives toward Regeneration.”

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3 Results

3.1 Role of EMMPRIN in tooth formation

Khaddam, Mayssam, Eric Huet, Benoît Vallée, Morad Bensidhoum, Dominique Le-

Denmat, Anna Filatova, Lucia Jimenez-Rojo, et al. 2014. “EMMPRIN/CD147

Deficiency Disturbs Ameloblast-Odontoblast Cross-Talk and Delays Enamel

Mineralization.” Bone

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3.1.1 Supplementary data

Figure 22 : EMMPRIN expression in the developing incisor of 3 month-old mice

Immunostaining with EMMPRIN antibody on sagittal section of the mandible shows

that the secretory ameloblasts, the stratum intermedium and odontoblasts are positive

for EMMPRIN (A and B). By contrast, no staining is observed in the post-secretory

ameloblast (C). Am: ameloblast; s-Am: secretory ameloblast; pos-Am: post-secretory

ameloblast; Od: odontoblast; D: dentin; pD: predentin; pm-E: premature enamel; Si:

stratum intermedium; fm: forming matrix. From (Khaddam et al. 2014)

70

Figure 23 : KLK-4 expression in tooth germs of EMMPRIN KO mice when compared

with WT. For mRNA expression, a 33 % increase is observed by qRT-PCR in KO mice.

KLK-4 activity is hardly detectable by casein zymography (with 20 mM EDTA in the

incubation buffer to inhibit MMP activity). No activity is seen for recombinant MMP-

20. From (Khaddam et al. 2014)

Supplementary Table 1 : PCR gene primers and reference. From (Khaddam et al. 2014).

71

Figure 24 : SEM observation of 3 month-old mouse mandible sections. At M1 level, no

difference in the morphology of either the bone or the teeth is detected between WT and

KO mice (A, B). Both dentin (E, F) and enamel appear normal and the enamel prisms

are normally constituted (C, D). From (Khaddam et al. 2014).

72

3.1.2 Supplementary results

3.1.2.1 Basement membrane degradation - Transmission electron microscopy

Basement membrane degradation is delayed in the molar germs of EMMPRIN KO

when compared to WT mice

3.1.2.1.1 Materials and methods

Mandibles of post-natal day 1 mice (3 litter-mate mice per group) were analyzed by

conventional transmission electron microscopy (TEM). Heads were fixed in 2% (w/v)

glutaraldehyde in 0.15 M cacodylate buffer, pH 7.4, overnight at 4°C. After post-fixation in

2% OsO4 for 1 h and dehydration in graded ethanol series at 4°C, the samples were

embedded in Epon 812 (Fluka). Semi-thin sections were stained with toluidine blue and

fuchsine. After washing the sections were dried and mounted in Eukitt. Ultrathin sections

were stained with uranyl acetate and lead citrate and were examined with a JEOL 1011

electron microscope.

3.1.2.1.2 Results

To explore EMMPRIN role in mediating direct ameloblast-odontoblast interactions, we

performed transmission electron microscopy analysis on the molar germs of new born mice.

TEM examination of M1 and M2 germs allowed the observation of the cells located at the tip

of the cusps which corresponds to the higher differentiation stage (Figure 25). On M2 germs,

the cell polarization observed in the WT (Figure 25.A) was not visible in the KO in either the

ameloblasts and the odontoblast layer (Figure 25.B). The basement membrane, which

separates the pre-ameloblast from the pre-odontoblast compartments, was already partially

degraded in the WT allowing for direct cell interactions between the two cellular

compartments (Figure 25.C), whereas it was still continuous in the KO (Figure 25.D),

suggesting that the differentiation process was delayed in EMMPRIN KO germs. However at

a later stage of the development (M1 germ), cells were fully polarized with a palisade

organization in both mice (Figure 25.E, F). The basement membrane at the tip of the cusp

was no longer detectable in either mice model (Figure 25.G,H). Dentin matrix was actively

secreted and mineralized although at a more advanced stage in the WT and mineralizing

enamel was already detectable (Figure 25.G).

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Figure 25: TEM analysis was performed on M1 and M2 germs of new born mice. In the

KO M2 germs, a cell polarization delay is observed in both pre-ameloblasts and pre-

odontoblasts localized at the tip of the cusps (b). In the WT, well-organized ameloblast

and odontoblast palisades are seen, with a basal localization of the nuclei and long cell

processes (arrow-heads) (a), whereas in the KO, cells are seen proliferating with

centrally localized nucleus (b). At higher magnification, the basement membrane (black

arrows) is partially degraded in WT (white arrows) (c), but appears still intact in the

KO (d). In M1 germ, the basement membrane which can no longer be detected in the

WT (e) is partially degraded in the KO (arrow) (f). Dentin matrix (black arrow-heads)

is secreted in both mice models (e-f-g-h) but at a higher rate in the WT (e) where a

greater amount of fibrillated collagen is seen associated with hydroxyapatite crystals

(white arrow heads). In addition, mineralizing enamel matrix can already be observed

at the secreting pole of WT ameloblasts localized at the tip of the cusp (g) but is not

detectable in the KO (h). pam: pre-ameloblast; pod: pre-odontoblast; am: ameloblast;

od: odontoblast; fde: forming dentin; fen: forming enamel. (Own data).

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3.1.2.2 EMMPRIN expression in the molar germ of mouse embryo

Figure 26: EMMPRIN expression in the first molar of mouse embryo.

Immunoreactivity (IR) for EMMPRIN in paraffin sections of mouse embryo tooth germ

tissue at 16 day and 17 day (cap stage). Inner enamel epithelium cells show EMMPRIN

IR, this IR in the buccal side is stronger than in the lingual side of the molar germ. Iee:

innerenamel epithelium; dp: dental pulp; bs: buccal side; ls: lingual side. (own data)

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3.1.2.3 Alveolar bone phenotype

3.1.2.3.1 Materials and methods

In order to explore the alveolar bone phenotype in EMMPRIN -/- mice, Half mandibles (n=3

age-matched mice per group) were subjected to a desktop micro-CT, (Skyscan 1172,

Skyscan, Aartselaar, Belgium). The micro-CT settings were used as follows: 9 μm resolution,

voltage 80 kV; current 100 μA; exposure time 400 ms; 180° rotation; rotation step 0.4 degree;

frame averaging 4. The scanning time was approximately 4 hours/sample. A total of 1700

native slice frames per sample were reconstructed using NRecon software (Skyscan,

Belgium). Tridimensional images were acquired with an isotropic voxel size of 9.92 μm. Full

3D high-resolution raw data are obtained by rotating both the X-ray source and the flat panel

detector 360° around the sample.

Bone volume rendering was measured using the open-source OsiriX imaging software

(v3.7.1, distributed under LGPL license, Dr A. Rosset, Geneva, Switzerland) from 2D

images.

The microarchitecture of alveolar bone of left mandible was studied. The ventral limit of the

volume of interest (VOI) was located at the first section containing the mesial root of the first

left molar; the dorsal limit was located 100 sections after, at the level of the alveolar ridge

and the buccal surface of the bone jaw (Figure 27). The VOI was designed by drawing

interactively polygons on the 2D sections. Several polygons were needed to be drawn (e.g. on

the first section, several at the middle, and on the final section) using the free hand tool with

“CT analyzer” software (Skyscan, release 1.13.5.1, Kontich, Belgium). The interpolated VOI

comprised only basal/alveolar bone. A simple global thresholding was determined

interactively to eliminate background noise and to select bone.

The parameters analyzed were: Bone volume fraction BV/TV (%), trabecular thickness

Tb.Th (mm), trabecular number Tb.N (1/mm), and trabecular separation Tb.Sp (mm).

Numerical variables were expressed as mean ± standard deviation.

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Figure 27: Alveolar bone density.

3.1.2.3.2 Results

3.1.2.3.2.1 Bone density

Bone density is presented by the percentage of space occupied by the spongy trabecular bone

in the volume of interest (VOI). It was calculated by measuring the ratio between the Percent

of trabecular bone volume and bone volume (BV / TV). The BV / TV in +/+ and -/-

EMMPRIN mice were almost the same (Figure 28).

Figure 28: Percent of bone volume in VOI.

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3.1.2.3.2.2 Trabecular bone characters

Trabecular bone characters are presented by measuring the following:

Thickness of bone trabeculae (Tb.Th) in the VOI.

The Tb.Th in +/+ and +/- EMMPRIN mice were almost the same (Figure 29).

Figure 29: Trabecular bone thickness in VOI.

The number of bone trabeculae (Tb.N) in the VOI.

The Tb.N in -/- EMMPRIN mice were about the same that of +/+ mice (Figure 30).

Figure 30: Trabecular number in VOI.

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The separation between bone trabeculae (Tb.Sp) in the VOI.

The Tb.Sp in the -/- EMMPRIN mice were about the same that of +/+ (Figure 31).

Figure 31: Trabecular separation in VOI.

79

3.2 Role of EMMPRIN in pulp-dentin regeneration

3.2.1 Background and project aim

EMMPRIN has been shown to be involved in the repair process of different injured tissues.

Indeed, the role of EMMPRIN in wound healing through MMP induction and increase in

myofibroblast contractile activity has been established (Gabison, Mourah, et al. 2005; Huet,

Vallée, et al. 2008). Therefore, the aim of this project was to investigate EMMPRIN role in

the pulp dentin repair process by comparing the healing of injured pulps of EMMPRIN KO

and WT mice.

3.2.2 Materials and methods

Twelve young adult mice (3 month-old) were used (6 WT mice and 6 EMMPRIN KO mice;

ethical agreement Animal Care Committee of the University Paris Descartes

CEEA34.CC.016.11). Following anaesthesia by intra-peritoneal injection of 2,2,2

tribromoethanol 2-methyl 2-butanol (Avertine®- Sigma Aldrich Germany) (0,017ml/g), a

small cavity was prepared with a carbide bur (Dia 0,04mm) (Komet- France) on the acclusal

aspect of the first upper left and right molars, in the centre of the tooth according to the

mesio-distal plane until the pulp was visible through the transparency of the dentine floor of

the cavity. A pulp exposure was mechanically done using an endodontic hand file of 0.15mm

diameter with a 4% taper (C+file®, Dentsply-Maillefer France). Pulp capping was performed

using Biodentine cement (septodont France) following the manufacturer’s recommendations.

Using the tip of a probe, Biodentine was placed in contact with the pulp, and slightly

condensed with a sterile paper point (XX-Fine, Henry Schein, France). Then, the cavity was

sealed with glass ionomer cement (GIC). Animals were placed in individual cages until they

recovered from anesthesia, and ibuprofen (0.06mg/g/day) was added in their drinking water

for 72 hours. Treated animals were sacrificed at increasing time points following the clinical

procedure, as follows:

- Six animals at 1 week post-operatively

- Six animals at 4 weeks post-operatively

Following removal of most of the soft tissues, heads of animals were immersed in 4%

para-formaldehyde (PFA) (Sigma) overnight at 4°C. Before demineralization prior to

histological analysis, samples were examined by micro-Ct at the same parameters

80

previously reported (page 75, materials and methods.). Micro-Ct data were analyzed

using the Osirix software and then Ct-analysis software.

3.2.3 Results

3.2.3.1 Micro-CT

In order to explore the tooth reparation, we performed Micro-CT on the maxilla of the treated

WT and KO mice (Figure 32).

Figure 32: Mouse first upper molar after 7 and 28 days of capping with Biodentine. 7

days post operatively, dentin formation was detected in +/+ and -/- EMMPRIN mice

(A,C), but it was more in -/- (brown arrow C) than in +/+ (brown arrow A). 28 days

post operatively, dentine bridge was visible, but it was more continuous in -/- (arrow in

D) than in +/+ (arrow in B) where it was not continued. e: enamel; d: dentin; GIC: glass

ionomer cement; red *: Biodentine.

81

Improved dentin repair is seen in KO when compared with WT both at Day 7 (P= 0.007 S)

and day 28 (P= 0.157 NS) (Figure 33). This data must now be supported by histological

analysis which are ongoing.

Figure 33: Percent of dentin volume in volume of interest VOI. Significant increase in

dentin density was detected in -/- EMMPRIN mice when compared with +/+ mice at 7

days post operatively.

82

3.3 Inhibition of MMP-3 and dentin matrix degradation

83

84

85

86

87

88

89

90

91

4 Discussion

Tooth development results from reciprocal inductive interactions between the

mesenchyme and the oral epithelium and proceeds through a series of well-defined stages

(Ruch, Karcher-Djuricic, and Gerber 1973; Slavkin 1974; Catón and Tucker 2009; Miletich

and Sharpe 2003; I Thesleff and Hurmerinta 1981; Mitsiadis and Luder 2011). Basement

membrane degradation allowing a direct contact between pre-ameloblasts and pre-

odontoblasts and their newly synthesized ECM appeared to be a crucial step of tooth

development (Meckel, Griebstein, and Neal 1965; Cevc et al. 1980; Zeichner-David et al.

1995). EMMPRIN, a membrane glycoprotein also named CD147, has been shown to play an

important role in the direct epithelial-mesenchymal interactions, as highlighted in the cancer

field (Toole 2003). The expression of EMMPRIN in the developing tooth germs has been

previously described (Schwab et al. 2007; Xie et al. 2010), increasing in the forming tooth

germ from E14 to P1 (Xie et al. 2010). In this thesis, we confirmed that EMMPRIN was first

expressed by pre-ameloblats and by the stratum intermedium at the early bell stage. At the

late bell stage, EMMRIN labeling decreased on ameloblasts actively secreting enamel

proteins, whereas it strongly increased on odontoblasts. Finally, this labeling disappeared in

postsecretory ameloblasts whose function is to mature the enamel matrix.

However, at the beginning of this thesis, the in vivo role of EMMPRIN in tooth

development and homeostasis was still unknown. By investigating mice KO for EMMPRIN,

we showed that EMMPRIN, through the induction of several MMPs, may orchestrate the

epithelial-mesenchymal cross-talk necessary for tooth formation, by enabling cleavage of the

basement membrane and thus direct cell-cell interactions. Indeed in our mice we showed a

delay in basement membrane degradation in KO mice when compared with WT. As a result,

we observed a delay in ameloblast differentiation, especially detectable on transmission

electron microscopy images (see Figure 25). As a consequence, MMP-3 and MMP-20

expression and activity were decreased and resulted in adults in decreased enamel volume

and subtle abnormalities at the DEJ in both molars and incisors. It is noteworthy that we

reported for the first time that EMMPRIN regulated the expression of MMP-20. As mice KO

for MMP-20 display early enamel shedding and severe tooth alterations, our data suggest

however that the quantity of MMP-20 expressed in the absence of EMMPRIN is sufficient to

allow correct enamel maturation. Therefore, enamel volume was decreased in EMMPRIN

KO mice but the maturation was rather-normal as indicated by nano-indentation experiments.

92

Surprisingly, in the tooth, EMMPRIN appeared to have no effect on MMP-2 and MMP-9

expression and activity. In several other processed such as corneal wound healing (Gabison et

al, 2009), the absence of EMMPRIN was shown to alter gelatinase expression and activity.

Our results therefore suggest that the action of EMMPRIN is organ-dependent.

Previous observations using Si-RNA experiments on mandibles in culture have

indicated that EMMPRIN was involved in tooth morphogenesis (Xie et al, 2010). In our

study, we showed that tooth phenotype was rather normal in EMMPRIN KO mice, which is

not consistent with these previous ex vivo observations. We therefore propose that the direct

effect of EMMPRIN on the epithelial-stromal interaction may be limited since it is only

allowed between basement membrane degradation allowing direct cell contact and before

calcified matrix deposition which constitutes cell barriers, hence limiting EMMPRIN’s action

(Figure 34).

Figure 34: Recapitulative schema proposing the role of EMMPRIN in tooth formation.

At early bell stage, EMMPRIN is expressed by pre-ameloblast (p-Am) and may

orchestrate basement membrane degradation (black line) to allow direct contact with

pre-odontoblast (p-Od), which is mandatory for the final cell differentiation. At

secretory stage, both secreting ameloblasts (s-Am) and odontoblasts (Od) highly express

EMMPRIN. This expression may enhance MMP-20 synthesis by ameloblasts allowing

for early enamel maturation. At the enamel maturation stage, post-secretory

ameloblasts (pos-Am) lose their EMMPRIN expression. The arrows indicate

EMMPRIN expression by cells. The red line schematizes the time window where a

direct effect of EMMPRIN is allowed by a direct cell contact. D: dentin; pD: predentin;

pm-E: premature enamel; E: enamel.

93

EMMPRIN has been shown to be involved in the repair process of different injured

tissues through MMP induction and increase in myofibroblast contractile activity (Gabison,

Mourah, et al. 2005; Huet, Vallée, et al. 2008). We investigate for a potential role of

EMMPRIN in the pulp dentin repair process by comparing the healing of injured pulps of

EMMPRIN KO and WT mice. The repair process seems to be improved in KO mice but

these preliminary data must be repeated and supported by histological analysis.

MMPs have been suggested to contribute to dentin caries progression and the

hypothesis that MMP inhibition would affect dentin caries progression is clinically

interesting. This hypothesis was sustained by in vivo studies in rat caries models where dentin

caries progression was delayed by intra-oral administration of chemical MMP inhibitors,

modified tetracylines and zoledronate (Sulkala et al. 2001; Tjäderhane et al. 1999). The

MMP-inhibitory effects of Grape-seed extracts (GSE) suggest that these natural substances

could be effective in inhibiting dentin caries progression. We therefore evaluated the capacity

of these natural agents incorporated in a mouthrinse to prevent the degradation of

demineralized dentin matrix by MMP-3. In this study, we selected MMP-3 because we have

previously shown that this enzyme was the only MMP among those tested (MMP-2, MMP-3

and MMP-9) that was able to degrade several NCPs (Boukpessi et al., 2008), known to be

associated to the collagen fibers in the dentin (Orsini et al., 2006). The removal of these

NCPs can then permit further matrix degradation by exposing the collagen fibers to more

collagen-specific MMPs such as collagenases and gelatinases (Malla et al., 2008) which are

also present in the dentin organic matrix and in the saliva (Tjaderhane et al., 1998).

Our results show that dentin pretreatment with the tested mouthrinse, and with its active

principles, inhibited the release by MMP-3 of several NCPs, namely decorin, biglycan and

DSP from the matrix and the disorganization along the dentinal tubules induced by MMP-3.

We therefore hypothesized that the inhibition of NCP cleavage by GSE may prevent further

matrix degradation by protecting the collagen fibers from collagen-specific MMPs such as

collagenases and gelatinases. Indeed, PGs were initially reported as the major substrates of

MMP-3. However, the situation may be more complex since PGs are bound to several other

proteins in the ECM (Qin et al., 2006), and once the degradation of the dentin ECM is

initiated, it may be more susceptible to further degradation by other proteases. Interestingly,

amine fluorides appear to have MMP inhibitory properties at a lesser degree than GSEs but at

94

a higher level than NaF. This information is clinically relevant, fluorides being recognized as

the most efficient tools for preventing dental caries. However, it requires further

investigations to be confirmed.

As general conclusion, proteases and their regulator such as EMMPRIN appear to have a

major role in the formation, pathologies and repair of the tooth. Therefore their understanding

opens several therapeutic issues, especially for the prevention and the treatment of dentin

caries.

95

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6 Annexe 1

Benjamin Salmon, Claire Bardet, Mayssam Khaddam, Jiar Naji, Benjamin R. Coyac,

Brigitte Baroukh, Franck Letourneur, Julie Lesieur, Franck Decup, Dominique Le Denmat,

Antonino Nicoletti, Anne Poliard, Peter S. Rowe, Eric Huet, Sibylle Opsahl Vital, Agne`s

Linglart, Marc D. McKee, Catherine Chaussain. 2013. “MEPE-Derived ASARM Peptide

Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in

Tooth Models of X-Linked Hypophosphatemia”

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Abstract

Role of EMMPRIN and MMPs in tooth development, dental caries and pulp-dentin regeneration

Tooth development is regulated by a series of reciprocal inductive signalings between the dental epithelium and

mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular

Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other

pathological processes and is expressed in developing teeth.

Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissues

formation. We demonstrated that EMMPRIN deficiency results in decreased in MMP-3 and MMP-20 expressions,

delayed in basement membrane degradation in tooth germ, delayed in enamel formation well distinguishable in

incisor, and in decreased enamel volume and thickness but normal maturation. These results indicate that

EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the

expression of MMPs.

Then we tried to investigate the potential role of EMMPRIN in the pulp dentin repair process by comparing the

healing of injured pulps of EMMPRIN KO and WT mice.

Finally, we evaluated the capacity of grape-seed extracts (known to be natural inhibitors of MMPs and used in

new daily mouthrinse) to prevent the degradation of human demineralized dentin matrix by MMP-3.

KEY WORDS: TOOTH FORMATION, MMPS, CELL INTERACTION, ENAMEL PROTEINS

Résumé

Rôle d'EMMPRIN et MMPS dans le développement dentaire, la carie dentaire et la régénération

pulpo-dentinaire

Le développement dentaire est orchestré par une série de signalisations inductives réciproques entre l'épithélium

dentaire et le mésenchyme, qui conduit à la formation de la dentine et de l'émail. EMMPRIN/CD147 est un

INducteur des MetalloPRoteinases de la Matrice Extracellulaire (MMPs) qui régule les interactions épithélio-

mésenchymateuses dans le cancer et d'autres processus pathologiques et est exprimé lors du développement

dentaire.

Ainsi, nous avons utilisé des souris KO pour EMMPRIN pour déterminer le rôle d'EMMPRIN dans la formation

des tissus dentaires. Nous avons démontré que l’absence d’EMMPRIN conduisait dans le germe dentaire à une

diminution de l’expression de MMP-3 et de MMP-20, à un retard de la dégradation de la membrane basale, à un

retard de la formation de l’émail bien visible dans l'incisive à croissance continue, à une diminution du volume et

de l'épaisseur d'émail, mais à une maturation amélaire normale. Ces résultats indiquent qu'EMMPRIN est

impliqué dans le dialogue épithélio-mésenchymateuse pendant le développement dentaire, principalement par la

régulation de l'expression de certaines MMPS.

Nous avons ensuite essayé d'évaluer le rôle potentiel d'EMMPRIN dans le processus de réparation dentaire en

comparant la cicatrisation de blessures pulpaires des souris KO pour EMMPRIN à des souris WT.

Enfin, dans un souci de transfert vers la clinique, nous avons évalué la capacité d’extraits de pépin de raisin

(connu pour être des inhibiteurs naturels de MMPs) à empêcher la dégradation de la matrice dentinaire humaine

déminéralisée et traitée par MMP-3.

MOTS CLÉS: FORMATION DE LA DENT, MMPS, INTERACTION CELLULAIRE, PROTÉINES DE

L’ÉMAIL.

143

Abstract

Role of EMMPRIN and MMPs in tooth development, dental caries and pulp-dentin regeneration

Tooth development is regulated by a series of reciprocal inductive signalings between the dental epithelium and

mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular

Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other

pathological processes and is expressed in developing teeth.

Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissues

formation. We demonstrated that EMMPRIN deficiency results in decreased in MMP-3 and MMP-20 expressions,

delayed in basement membrane degradation in tooth germ, delayed in enamel formation well distinguishable in

incisor, and in decreased enamel volume and thickness but normal maturation. These results indicate that

EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the

expression of MMPs.

Then we tried to investigate the potential role of EMMPRIN in the pulp dentin repair process by comparing the

healing of injured pulps of EMMPRIN KO and WT mice.

Finally, we evaluated the capacity of grape-seed extracts (known to be natural inhibitors of MMPs and used in

new daily mouthrinse) to prevent the degradation of human demineralized dentin matrix by MMP-3.

KEY WORDS: TOOTH FORMATION, MMPS, CELL INTERACTION, ENAMEL PROTEINS

Résumé

Rôle d'EMMPRIN et MMPS dans le développement dentaire, la carie dentaire et la régénération

pulpo-dentinaire

Le développement dentaire est orchestré par une série de signalisations inductives réciproques entre l'épithélium

dentaire et le mésenchyme, qui conduit à la formation de la dentine et de l'émail. EMMPRIN/CD147 est un

INducteur des MetalloPRoteinases de la Matrice Extracellulaire (MMPs) qui régule les interactions épithélio-

mésenchymateuses dans le cancer et d'autres processus pathologiques et est exprimé lors du développement

dentaire.

Ainsi, nous avons utilisé des souris KO pour EMMPRIN pour déterminer le rôle d'EMMPRIN dans la formation

des tissus dentaires. Nous avons démontré que l’absence d’EMMPRIN conduisait dans le germe dentaire à une

diminution de l’expression de MMP-3 et de MMP-20, à un retard de la dégradation de la membrane basale, à un

retard de la formation de l’émail bien visible dans l'incisive à croissance continue, à une diminution du volume et

de l'épaisseur d'émail, mais à une maturation amélaire normale. Ces résultats indiquent qu'EMMPRIN est

impliqué dans le dialogue épithélio-mésenchymateuse pendant le développement dentaire, principalement par la

régulation de l'expression de certaines MMPS.

Nous avons ensuite essayé d'évaluer le rôle potentiel d'EMMPRIN dans le processus de réparation dentaire en

comparant la cicatrisation de blessures pulpaires des souris KO pour EMMPRIN à des souris WT.

Enfin, dans un souci de transfert vers la clinique, nous avons évalué la capacité d’extraits de pépin de raisin

(connu pour être des inhibiteurs naturels de MMPs) à empêcher la dégradation de la matrice dentinaire humaine

déminéralisée et traitée par MMP-3.

MOTS CLÉS: FORMATION DE LA DENT, MMPS, INTERACTION CELLULAIRE, PROTÉINES DE

L’ÉMAIL.